Biofilms with immobilized cells in industrial fermentation are beneficial. Encased in extracellular polymeric substance, cells forming biofilms are regulated by various factors. Nitric oxide (NO), as a signaling molecule, recognized as quorum sensing molecule regulating microbe biofilm formation. Regulation mechanisms of NO on bacteria biofilm have been studied extensively and deeply, while on fungus are rarely studied. In this study, we observed that low concentration of NO enhanced S. cerevisiae biofilm formation. Transcriptional and proteomic analysis revealed that transcription factor MAC1 was activated in biofilm cells under NO treatment. Overexpressed MAC1 increased yeast biofilm formation bypassing regulating the expression level of FLO11. Increased copper and iron contents in NO treated and MAC1 overexpressed cells were not responsible for increased biofilm formation. Among six downstream genes of MAC1, overexpressed CTR1 contributed yeast biofilm formation. Moreover, MAC1 and CTR1 contributed to biofilm cells ethanol resistance resulting from enhanced biofilm. The role of CTR1 protein in yeast biofilm formation may result from its hydrophobic residues in N-terminal extracellular domain. These findings suggested a NO-mediated biofilm formation mechanism that NO regulated expression levels of CTR1 through activating its transcription factor MAC1, leading enhanced biofilm formation.