Yeast culture (YC) is widely used in dairy production to enhance milk yield and quality, yet effects vary due to differences in products, doses, and trial conditions. This meta-analysis evaluated the impact of YC supplementation on milk yield and composition in lactating Holstein cows, aiming to identify effective yeast culture types, dosages, and duration of use. A systematic search of PubMed, Web of Science, and CNKI for randomized controlled trials (RCTs) from 2000 to 2024 was conducted. Following PICOS criteria, 23 RCTs comprising 32 comparisons and over 3200 cows were included. Data were analyzed using RevMan 5.3 and Stata/MP 15.0 to compute standardized mean differences (SMD) and 95% confidence intervals with random-effects models. Subgroup and sensitivity analyses were performed. Results showed that YC supplementation significantly improved milk yield (SMD = 2.14), fat (SMD = 0.57), protein (SMD = 1.34), and lactose content (SMD = 0.61). Subgroup analysis revealed that supplementation with saccharomyces cerevisiae at a dosage of 10-50 g/d effectively increased milk yield during lactation 42-56 d. In contrast, during the lactation 21-30 d, different dosages of saccharomyces cerevisiae exerted differential effects on milk composition: supplementation at 60-120 g/d contributed to an increase in milk fat content, while supplementation at 10-50 g/d significantly enhanced milk protein level. Furthermore, lactose content was not significantly associated with the feeding period of saccharomyces cerevisiae; however, high-dose (>120 g/d) could significantly increase lactose content. Significant heterogeneity (I2 = 70.7-89.6%) was observed, largely due to strain and dose variations. In conclusion, YC effectively enhances milk production and composition, with optimal outcomes depending on yeast type, dose, and duration, providing evidence-based recommendations for targeted supplementation strategies.