To investigate the predictive value of the serum 25-hydroxyvitamin D (25-(OH) D) level combined with the homocysteine (Hcy) level of women with secondary infertility for their pregnancy situation after treatment. Methods: 161 women with secondary infertility from January 2022-August 2023 were selected in study group, and 161 normal pregnant women were selected in control group. The general data and the serum -(OH) D and Hcy levels of the women were compared between the two groups. The women in the study group were followed up for 6 months. According to the pregnancy situation after treatment, the women in the study group were divided into group A (women with clinical pregnancy) and group B (women without clinical pregnancy). Receiver operating characteristic (ROC) curve was used to analyze the predictive value of the serum 25-(OH) D and Hcy levels of the women in the study group for their pregnancy after treatment. Results: The serum Hcy level (10.50±5.57μmol/L) of the women in the study group was significantly higher than that (21.02±5.62μmol/L) of the women in the control group. The level of 25-(OH)D (18.31±6.34 ng/ml) of the women in the study group was significantly lower than that (21.02±5.62 ng/ml) of the women in the control group. The serum 25-(OH) D (19.84±6.20 ng/ml) level of the women in group A was significantly higher than that (15.52±6.60 ng/ml) of the women in group B, and the Hcy level (8.72±5.29μmol/L) of the women in group A was significantly lower than that (13.75±6.08μmol/L) of the women in group B (all P<0.05). The area under the curve of the 25-(OH)D level combined with the Hcy level of the women in the study group for predicting their pregnancy after treatment was 0.884, which was significantly higher than that (0.767) of the 25-(OH)D  level or that (0.842) of the Hcy level alone. The sensitivity and the specificity of the 25-(OH)D level combined with the Hcy level of the women in the study group for predicting their pregnancy after treatment were 82.5% and 80.8%. Conclusion: The serum 25-(OH) D3 level of the women with the secondary infertility is lowly expressed, and the serum Hcy level of the women is highly expressed. The combined detections of the serum 25-(OH) D3 level and the Hcy level has good predictive value for their pregnancy after treatment.