There is difference of opinion among biographers regarding the year of birth of Tulsidas. Many sources rely on Veni Madhav Das' account in the Mula Gosain Charita, which gives the year of Tulsidas' birth as Vikrami Samvat 1554 (1497 CE). These sources include Shivlal Pathak, popular editions of Ramcharitmanas (Gita Press, Naval Kishore Press and Venkateshvar Press), Edwin Greaves, Hanuman Prasad Poddar, Ramanand Sarasvati, Ayodhyanath Sharma, Ramchandra Shukla, Narayandas, and Rambhadracharya. A second group of biographers led by Sant Tulsi Sahib of Hathras and Sir George Grierson give the year as Vikram 1589 (1532 CE). These biographers include Ramkrishna Gopal Bhandarkar, Ramghulam Dwivedi, James Lochtefeld, Swami Sivananda and others. A third small group of authors which includes H. H. Wilson, Garse De Tasse and Krishnadatta Mishra gives the year as Vikram 1600 (1543 CE). The year 1497 appears in many current-day biographies in India and in popular culture. Biographers who disagree with this year argue that it makes the life span of Tulsidas equal 126 years, which in their opinion is unlikely if not impossible. In contrast, Ramchandra Shukla says that an age of 126 is not impossible for a Mahatma (great soul) like Tulsidas. The Government of India and provincial governments celebrated the 500th birth anniversary of Tulsidas in the year 2011 CE, according to the year of Tulsidas' birth in popular culture.

Ramacharitamanas (रामचरितमानस, 1574–1576), "The Mānasa lake brimming over with the exploits of Lord Rāma" is an Awadhi rendering of the Ramayana narrative. It is the longest and earliest work of Tulsidas, and draws from various sources including the Ramayana of Valmiki, the Adhyatma Ramayana, the Prasannaraghava and Hanuman Nataka. The work consists of around 12,800 lines divided into 1073 stanzas, which are groups of Chaupais separated by Dohas or Sorthas. It is divided into seven books (Kands) like the Ramayana of Valmiki, and is around one-third of the size of Valmiki's Ramayana. The work is composed in 18 metres which include ten Sanskrit metres (Anushtup, Shardulvikridit, Vasantatilaka, Vamshashta, Upajati, Pramanika, Malini, Sragdhara, Rathoddhata and Bhujangaprayata) and eight Prakrit metres (Soratha, Doha, Chaupai, Harigitika, Tribhangi, Chaupaiya, Trotaka and Tomara). It is popularly referred to as Tulsikrit Ramayana, literally The Ramayana composed by Tulsidas. The work has been acclaimed as "the living sum of Indian culture", "the tallest tree in the magic garden of medieval Indian poesy", "the greatest book of all devotional literature", "the Bible of Northern India", and "the best and most trustworthy guide to the popular living faith of its people."

Tulsidas himself has given only a few facts and hints about events of his life in various works. Till late nineteenth century, the two widely known ancient sources on Tulsidas' life were the Bhaktamal composed by Nabhadas between 1583 and 1639, and a commentary on Bhaktamal titled Bhaktirasbodhini composed by Priyadas in 1712. Nabhadas was a contemporary of Tulsidas and wrote a six-line stanza on Tulsidas describing him as an incarnation of Valmiki. Priyadas' work was composed around a hundred years after the death of Tulsidas and had eleven additional stanzas, describing seven miracles or spiritual experiences from the life of Tulsidas. During the 1920s, two more ancient biographies of Tulsidas were published based on old manuscripts – the Mula Gosain Charit composed by Veni Madhav Das in 1630 and the Gosain Charit composed by Dasanidas (also known as Bhavanidas) around 1770. Veni Madhav Das was a disciple and contemporary of Tulsidas and his work gave a new date for Tulsidas' birth. The work by Bhavanidas presented more narratives in greater detail as compared to the work by Priyadas. In the 1950s a fifth ancient account was published based on an old manuscript, the Gautam Chandrika composed by Krishnadatta Misra of Varanasi in 1624. Krishnadatta Misra's father was a close companion of Tulsidas. The accounts published later are not considered authentic by some modern scholars, whereas some other scholars have been unwilling to dismiss them. Together, these five works form a set of traditional biographies on which modern biographies of Tulsidas are based.

As per Priyadas' account, Tulsidas followed the instruction of Hanumana and started living in an Ashram at Ramghat in Chitrakuta. One day Tulsidas went to perform the Parikrama (circumambulation) of the Kamadgiri mountain. He saw two princes, one dark and the other fair, dressed in green robes pass by mounted on horsebacks. Tulsidas was enraptured at the sight, however he could not recognise them and took his eyes off them. Later Hanuman asked Tulsidas if he saw Rama and his brother Lakshmana on horses. Tulsidas was disappointed and repentful. Hanuman assured Tulsidas that he would have the sight of Rama once again the next morning. Tulsidas recalls this incident in a song of the Gitavali and laments how "his eyes turned his own enemies" by staying fixed to the ground and how everything happened in a trice. On the next morning, Wednesday, the new-moon day of Magha, Vikram 1607 (1551 CE) or 1620 (1564 CE) as per some sources, Rama again appeared to Tulsidas, this time as a child. Tulsidas was making sandalwood paste when a child came and asked for a sandalwood Tilaka (a religious mark on the forehead). This time Hanuman gave a hint to Tulsidas and he had a full view of Rama. Tulsidas was so charmed that he forgot about the sandalwood. Rama took the sandalwood paste and put a Tilaka himself on his forehead and Tulsidas' forehead before disappearing.

In Vikram 1628 (1572 CE), Tulsidas left Chitrakuta for Prayag where he stayed during the Magha Mela (the annual fair in January). Six days after the Mela ended, he had the Darshan of the sages Yajnavalkya and Bharadvaja under a banyan tree. In one of the four dialogues in the Ramcharitmanas, Yajnavalkya is the speaker and Bharadvaja the listener. Tulsidas describes the meeting between Yajnavalkya and Bharadvaja after a Magha Mela festival in the Ramcharitmanas, it is this meeting where Yajnavalkya narrates the Ramcharitmanas to Bharadvaja.

In the year Vikram 1631 (1575 CE), Tulsidas started composing the Ramcharitmanas in Ayodhya on Tuesday, Ramnavami day (ninth day of the bright half of the Chaitra month, which is the birthday of Rama). Tulsidas himself attests this date in the Ramcharitmanas. He composed the epic over two years, seven months and twenty-six days, and completed the work in Vikram 1633 (1577 CE) on the Vivaha Panchami day (fifth day of the bright half of the Margashirsha month, which commenrates the wedding of Rama and his wife Sita).

Several manuscripts of the Ramcharitmanas are claimed to have been written down by Tulsidas himself. Grierson wrote in the late nineteenth century, two copies of the epic were said to have existed in the poet's own handwriting. One manuscript was kept at Rajapur, of which only the Ayodhyakand is left now, which bears marks of water. A legend goes that the manuscript was stolen and thrown into Yamuna river when the thief was being pursued, and only the second book of the epic could be rescued. Grierson wrote that the other copy was at Malihabad in Lucknow district, of which only one leaf was missing. Another manuscript of the Ayodhyakanda claimed to be in the poet's own hand exists at Soron in Etah district, one of the places claimed to be Tulsidas' birthplace. One manuscript of Balakanda, dated Samvat 1661, nineteen years before the poet's death, claimed to be corrected by Tulsidas, is at Ayodhya. Some other ancient manuscripts are found in Varanasi, including one in possession of the Maharaja of Benares that was written in Vikram 1704 (1647), twenty-four years after the death of Tulsidas.

Around Vikram 1664 (1607 CE), Tulsidas was afflicted by acute pain all over his body, especially in his arms. He then composed the Hanuman Bahuk, where he describes his bodily pain and suffering in several stanzas. He was relieved of his pain after this composition. Later he was also afflicted by Bartod boils (Hindi: बरतोड़, furuncles caused by pulling out of the hair), which may have been the cause of his death.

The Vinaypatrika is considered as the last compositions of Tulsidas, believed to be written when Kali Yuga started troubling him. In this work of 279 stanzas, he beseeches Rama to give him Bhakti ("devotion"), and to accept his petition. Tulsidas attests in the last stanza of Vinaypatrika that Rama himself signed the manuscript of the work. The 45th stanza of the Vinaypatrika is sung as the evening Aarti by many Hindus.

Specifically about his poetry, Tulsidas has been called the "emperor of the metaphor" and one who excels in similes by several critics. The Hindi poet Ayodhyasingh Upadhyay 'Hariaudh' said of Tulsidas –