The quality of wort used in beer fermentation directly influences beer quality. A metabolomics approach was adopted to investigate how different saccharification processes affect fermented beer quality. By analyzing the differences in wort composition under different saccharification conditions, the impact of wort from these processes on the metabolic pathways of Saccharomyces cerevisiae was elucidated. Principal component analysis and partial least squares discriminant analysis of the data from mass spectrometry revealed that wort from different saccharification processes affected the metabolism of amino acids, organic acids, lipids, and fatty acids in S. cerevisiae. This mainly involves arginine biosynthesis; biosynthesis of phenylalanine, tyrosine, and tryptophan; biosynthesis of valine, leucine, and isoleucine; metabolism of glycine, serine, and threonine; D-amino acid metabolism; lysine degradation; and fatty acid biosynthesis. These pathways significantly influence beer flavor, offering theoretical insights for regulating flavor compounds in beer.