To explore the levels of serum vitamin D and soluble vascular endothelial growth factor receptor-1 (sFlt-1) of women with multiple uterine fibroids, and to study their diagnostic value for multiple uterine fibroids. Methods: 60 women with multiple uterine fibroids were selected in group A, 60 women with single uterine fibroids were selected in group B, and 60 healthy women who received physical examination were selected in group C from February 2018 to October 2020. The women in group A further divided in group A1 (women with uterine fibroids diameter <3cm) and group A2 (women with uterine fibroids diameter ≥3cm). The women in group B were further divided in group B1 (women with uterine fibroids diameter <3cm) and group B2 (women with uterine fibroids diameter ≥3cm). The serum vitamin D and sFlt-1 levels of the women were detected and were compared among these groups. Receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of the serum vitamin D and sFlt-1 levels of the women for their multiple uterine fibroids. Results: The serum vitamin D level of the women in group C, group B, and group A had decreased successively, and which of the women in group B was significantly lower than that of the women in group C. The serum sFLT-1 level of the women in group C, group B, and group A had increased successively, and which of the women in group B was significantly higher than that of the women in group C. The serum vitamin D level of the women in group A2 and group B2 was significantly lower than that of the women in group A1 and group B1, and the serum sFLT-1 level of A2 group B2 was significantly higher than that of group A1 and group B1. The serum vitamin D level of the women in group A was significantly lower than that of the women in group B, and the serum sFLT-1 level of the women in group A was significantly higher than that of the women in group B (all P<0.05). ROC curve analysis showed that the AUC of vitamin D level of the women for diagnosing their multiple uterine fibroids was 0.811, and the sensitivity and the specificity of which were 65.8% and 78.4% with the cut-off value of 16.23ng/ml. The AUC of SFLT-1 level of the women for diagnosing their multiple uterine fibroids was 0.875, and the sensitivity and specificity of which were 88.3% and 79.6% with the cut-off value of 1.31ng/ml. The AUC, the sensitivity, and the specificity of the combination of the levels of vitamin D and sFlt-1 of the women for diagnosing their multiple uterine fibroids were 0.965, 95.9%, and 97.4%, respectively. Conclusion: Both the vitamin D deficiency and the increase of serum sFlt-1 level of the women may be closely related to their occurrence and development of multiple uterine fibroids. The combination of the levels of vitamin D and sFlt-1 of the women for diagnosing their early multiple uterine fibroids has higher efficacy.