Purpose Previous studies suggest that homocysteine (Hcy) may be involved in the pathogenesis of bipolar disorder (BD) and major depressive disorder (MDD) by influencing glutamatergic transmission, inflammation, and other mechanisms. There are no established biomarkers to distinguish BD from MDD. This study aims to compare Hcy levels between BD and MDD. Patients and Methods We collected medical records of patients aged 14–75 admitted to the hospital from January 1 to July 1, 2022 with a discharge diagnosis of MDD or BD, including all examinations of patients at admission (acute phase) and discharge (non-acute phase). We measured Hcy levels in healthy controls (HC). Results The analysis included 104 patients with MDD, 103 patients with BD, and 80 HC. Hcy levels were higher in the MDD and BD group than in the HC group and higher in the BD group than in the MDD group, both in the acute and non-acute phases (all P < 0.05). There was no significant difference in Hcy levels between the psychotropic medication users and non-users in the BD or MDD group (all P > 0.05). Multivariate logistic regression analysis only for the MDD and BD group indicated that the likelihood of BD diagnosis was significantly associated with Hcy levels (in the acute phase: OR = 1.052, P = 0.016; in the non-acute phase: OR = 1.101, P < 0.001) after controlling for gender, age, and metabolic indicators. Conclusion Our study suggests that Hcy levels were elevated in MDD and BD patients and were higher in BD patients than in MDD patients, which provides evidence for a possible relationship between one-carbon metabolism and the pathogenesis of BD. Besides, Hcy may be one of the potential biomarkers to distinguish BD from MDD.