Background <p>Sarcopenia, characterized by progressive muscle mass and strength decline, poses a significant health challenge among older adults, especially those with chronic diseases. Our study aims to evaluate the combined diagnostic potential of irisin, 25(OH)D3, and albumin for sarcopenia in older patients with chronic conditions.</p> Methods <p>A cohort of 393 older patients with chronic diseases, including 117 diagnosed with sarcopenia were included. Fasting blood samples were collected, and serum biomarkers (25(OH)D3, albumin, and irisin) were measured using automated biochemical analyzers and enzyme-linked immunosorbent assay to evaluate nutritional and muscle-related parameters.</p> Results <p>The prevalence of sarcopenia was higher among patients aged 80 or older compared to younger age groups in our study population. Strong associations were observed between sarcopenia and osteoporosis, tumors, and risk of malnutrition. Serum irisin, 25(OH)D3, and albumin levels were significantly lower in sarcopenic patients. Individual biomarkers displayed diagnostic potential, and a combined biomarker test showed superior accuracy. Multivariate logistic regression identified age, osteoporosis, malnutrition, and fatigue as independent risk factors, while higher serum biomarker levels correlated with reduced sarcopenia risk. Positive correlations were observed between serum biomarkers and sarcopenia severity indicators.</p> Conclusions <p>This study highlights the potential of irisin, 25(OH)D3, and albumin as diagnostic and prognostic tools for sarcopenia in older patients with chronic diseases, contributing to early detection and intervention strategies to enhance their quality of life.</p>