Evidences have shown that both LRFN5 and OLFM4 can regulate neural development and synaptic function. Recent genome-wide association studies on major depressive disorder (MDD) have implicated LRFN5 and OLFM4, but their expressions and roles in MDD are still completely unclear. Here, we examined serum concentrations of LRFN5 and OLFM4 in 99 drug-naive MDD patients, 90 drug-treatment MDD patients, and 81 healthy controls (HCs) using ELISA methods. The results showed that both LRFN5 and OLFM4 levels were considerably higher in MDD patients compared to HCs, and were significantly lower in drug-treatment MDD patients than in drug-naive MDD patients. However, there were no significant differences between MDD patients who received a single antidepressant and a combination of antidepressants. Pearson correlation analysis showed that they were associated with the clinical data, including Hamilton Depression Scale score, age, duration of illness, fasting blood glucose, serum lipids, and hepatic, renal, or thyroid function. Moreover, these two molecules both yielded fairly excellent diagnostic performance in diagnosing MDD. In addition, a combination of LRFN5 and OLFM4 demonstrated a better diagnostic effectiveness, with an area under curve of 0.974 in the training set and 0.975 in the testing set. Taken together, our data suggest that LRFN5 and OLFM4 may be implicated in the pathophysiology of MDD and the combination of LRFN5 and OLFM4 may offer a diagnostic biomarker panel for MDD.