Abstract: [Objective] This study aims to explore the application value of homocysteine (Hcy) levels in the serum of patients with non-traumatic osteonecrosis of the femoral head (NONFH) for early clinical diagnosis of the disease.[Methods] The study included 73 NONFH patients treated at Linyi People""s Hospital from January to October 2023 as the research group, and 68 healthy individuals matched by gender and age in the same period as the control group. The levels of Hcy in participants"" serum were measured using Enzyme-Linked Immunosorbent Assay (ELISA) and compared with clinical indicators such as etiology, affected side, degree of collapse, and ARCO staging. Additionally, the study analyzed the correlation between Hcy levels and both Visual Analogue Scale (VAS) scores and Harris Hip Scores.[Results] The study found that the serum Hcy levels in NONFH patients were significantly higher than those in the control group (P<0.05). There was no significant statistical difference in Hcy levels between patients with different etiologies and smoking habits; however, bilateral involvement patients showed significantly higher Hcy levels than unilateral involvement (P<0.05). Hcy levels were significantly higher in patients with femoral head collapse than those without (P<0.05). With the progression of ARCO staging, serum Hcy levels showed an increasing trend. Serum Hcy concentrations were positively correlated with VAS scores and ARCO staging but negatively correlated with Harris scores. The ROC curve indicated a sensitivity of 67.1% and a specificity of 85.3% for serum Hcy levels in diagnosing femoral head collapse, with an area under the curve (AUC) of 0.818.[Conclusion] The serum Hcy levels in NONFH patients are significantly elevated and positively correlated with the severity of the disease, indicating that Hcy is a potential diagnostic biomarker for NONFH.