This study was designed to validate thrombospondin 1 (TSP-1), vascular endothelial-cadherin complex (VE-cad), insulin-like growth factor 2 (IGF-2), and amyloid precursor protein (APP) and assess their diagnostic value in ventricular septal defect (VSD). We investigated the serum levels of TSP-1, VE-cad, IGF-2, and APP by enzyme-linked immunosorbent assay in a hospital-based case–control study that included 40 VSD children and 40 healthy controls. Logistic regression analysis was applied to evaluate the relationship of the proteins and VSD, and receiver operating characteristic (ROC) curve was used to assess the diagnostic value of the significant proteins. The serum levels of TSP-1, VE-cad, and IGF-2 were significantly higher in VSD patients than those in healthy controls (p < 0.05). Multivariate logistic regression analysis demonstrated that high levels of TSP-1, VE-cad, and IGF-2 were significantly associated with an increased risk of VSD [TSP-1 (OR 26.91, 95% CI 6.60–72.66, p < 0.001), VE-cad (OR 11.91, 95% CI 3.90–36.36, p < 0.001), IGF-2 (OR 3.25, 95% CI 1.25–8.43, p = 0.015)]. Areas under the ROC curve for TSP-1, VE-cad, and IGF-2 were 0.985, 0.838, and 0.658, respectively. These data demonstrated that TSP-1, VE-cad, and IGF-2 were significantly associated with risk of VSD and manifested diagnostic values, which may provide new evidence for understanding the etiology and promote the early diagnosis and prevention of VSD.