Introduction: Patients with Chronic Kidney Disease (CKD) undergoing regular hemodialysis experience various metabolic changes, including premature aging marked by increased prevalence of frailty. Beta-2 microglobulin (B2M), a uremic toxin whose concentration significantly increases in hemodialysis patients, has emerged as a potential biomarker of frailty. Previous evidence suggests potential link between B2M and frailty in older adults. However, data on its relationship with frailty in hemodialysis patients remains limited. Purpose: To determine the relationship between B2M concentration and frailty in hemodialysis patients. Patients and Methods: This is a cross-sectional study utilizing primary data from hemodialysis patients at Rumah Sakit Cipto Mangunkusumo (RSCM), employing a total sampling method. Beta-2 microglobulin was measured using the Enzyme-Linked Fluorescent Assay (ELFA) method. Frailty was assessed using the Frailty Index 40-item. Medical history was obtained from medical records and interviews. Chi-square tests were performed to determine the relationship between B2M and frailty. Multivariate analysis was conducted to determine variables that affect frailty. Results: A total of 79 subjects participated in the study. The median B2M concentration was 32.8 (IQR 29.8–36.77). Higher B2M concentration showed a trend toward increased frailty prevalence (PR of 4.83, 95% CI 0.69–33.81, p = 0.113). The final multivariate analysis showed that sarcopenia (PR 5.37; 95% CI 2.88–10.04) was strongly and consistently associated with frailty prevalence. Conclusion: Higher B2M showed a trend towards increased frailty prevalence; however, this association is not statistically significant. Sarcopenia is a significant factor influencing the prevalence of frailty in hemodialysis patients. © 2025 Nugroho et al.