Four kinds of worts with different nitrogen compositions were used to examine their effects on fermentation performance of brewer's yeast. The absorption pattern of peptides with different molecular weights (Mw) in yeast cells during wort fermentation was also investigated. Results showed that both the nitrogen composition and level had significant impacts on the yeast biomass accumulation, ethanol production, and free amino nitrogen and sugars consumption rates. Worts supplemented with wheat gluten hydrolysates increased 11.5% of the biomass, 5.9% of fermentability, and 0.6% of ethanol content and decreased 25.6% of residual sugar content during wort fermentation. Moreover, yeast cells assimilated peptides with various Mw differently during fermentation. Peptides with Mw below 1 kDa decreased quickly, and the rate of assimilation was more than 50% at the end of fermentation, while those with Mw above 10 kDa almost could not be assimilated by yeast. All these results further indicated that the level and composition of wort nitrogen had significant impacts on the growth and fermentation performances of brewer's yeast, and peptides with Mw below 1 kDa were one of preferred nitrogen sources for brewer's yeast.