Cell line development of Chinese hamster ovary (CHO) faces persistent challenges in selecting high-producing clones, particularly for complex therapeutic proteins requiring multigene co-expression. To address this, a novel tyrosine-based selection system incorporating three selection markers was developed based on three crucial enzymes in tyrosine biosynthesis: pterin-4α carbinolamine dehydratase 1 (PCBD1), phenylalanine hydroxylase (PAH), and quinoid dihydropteridine reductase (QDPR). Following systematic evaluation of sgRNA editing efficiency, a two-step knockout strategy was implemented: initial PCBD1 ablation followed by concurrent PAH/QDPR disruption, generating a triple-knockout CHO-Tyr-ko cell line. This engineered chassis exhibited strict tyrosine auxotrophy that was specifically rescued by pcbd1/ pah/ qdpr co-expression. The coupling of triple-fluorescent reporters ( mCherry/ copGFP/ mTagBFP2) with three selection markers ( pcbd1/ pah/ qdpr) via expression cassettes in tyrosine-based selection system demonstrated enhanced reporter expression levels, improved population homogeneity, and achieved 97.49% enrichment of triple-positive cells under tyrosine deprivation. Moreover, combined with optimized tyrosine supplement strategies, recombinant CHO-Tyr-ko-H/L/L cells selected in tyrosine-free medium attained a monoclonal antibody (mAb) titer of 0.35 g/l in fed-batch culture and 1.60 g/l in perfusion culture. In conclusion, the novel tyrosine-based selection system, featuring three selection markers under unified selection pressure, provides an alternative for recombinant protein expression and paves a new avenue for multigene co-regulation engineering in CHO cells.