<p>β-galactosidase (EC 3.2.1.23), a pivotal enzyme in lactose degradation, is widely distributed across numerous bacterial species. This study aimed to develop degenerate primers with high specificity and universality, using the β-galactosidase gene as a molecular marker to identify diverse lactose-degrading bacteria in environmental samples. High-quality β-galactosidase gene sequences belonging to glycoside hydrolase family 2 were retrieved from the NCBI and CAZY databases for sequence alignment and phylogenetic analysis. Through extensive screening and optimization of full-length sequences, a pair of primers was successfully designed for detecting environmental β-galactosidase genes. The newly developed primers were validated using four types of DNA samples, revealing the diversity of lactose-degrading bacterial communities: Human feces (2 phyla, 5 genera), Rat feces (3 phyla, 3 genera), Mouse feces (2 phyla, 6 genera), and soil (6 phyla, 13 genera). By optimizing sequences of representative strains based on the full-length target gene, this study successfully developed novel degenerate primers, providing a reference strategy for addressing the challenge of primer design for highly variable gene families. Additionally, a new molecular probe targeting the β-galactosidase gene was developed to identify diverse lactose-degrading bacteria in environmental samples. To the best of our knowledge, this is the first report of universal primers targeting the β-galactosidase gene for detecting lactose-degrading microbial communities in environmental samples.</p>