Blahoslav was born in Přerov, Moravia. He studied theory under Listenius and Hermann Finck at University of Wittenberg from 1544. At Wittenberg he became acquainted with Martin Luther, and he was also acquainted with Philipp Melanchthon. After a short period at Mladá Boleslav (1548–9) he continued his education at Königsberg and Basle. He was a fine linguist who strove to preserve the purity of his native tongue and succeeded in bridging the gulf between Christianity and humanism. He was ordained at Mladá Boleslav in 1553, and became a bishop of the Fraternity of Czech (or Moravian) Brethren in 1557. In the following year he established himself at Ivančice, where before long he installed a printing press. Towards the end of his life he moved to Moravský Krumlov, where he died, aged 48.
His treatise Musica: to gest knjžka zpěwákům, published in Olomouc in 1558 (ed. and Eng. trans. in Sovík), is believed to be the first on music theory in the Czech language, but its information is derived from the writings of Listenius, Finck, Ornithoparchus and Coclico. Blahoslav wrote two entirely new sections for the second edition giving critical and practical advice to singers and choirmasters, and guidance to composers of hymns: he emphasized the need for the musical rhythm to correspond with the časomíra system of prosody (i.e. the alternation of long and short syllables) of the verses. He was the chief editor of the Pisně duchowni ewangelistské (1561), known as the Szamotuły Kancionál, which comprised 735 hymn texts, 52 of them by Blahoslav, and more than 450 tunes, including a number drawn from secular sources and eight which he may have composed himself. Blahoslav has been greatly esteemed for his Czech translation of the New Testament (1568; ed. J. Konopásek, Prague, 1931–2), which was the initial step in the preparation of the celebrated Bible of Kralice (1588). Blahoslav's work had some influence on Jan Amos Komenský.