Clinical application value was investigated of transvaginal color Doppler ultrasound (TV-CDS) combined with serum tumor markers carbohydrate antigen 125 (CA125), vascular endothelial growth factor (VEGF) and osteopontin (OPN) in the diagnosis of ovarian cancer (OC). One hundred and six patients with OC [malignant tumor group (MTG)] and fifty patients with benign ovarian diseases [benign control group (BCG)] were selected. Both groups of patients underwent TV-CDS examination. The lesion morphology and internal structure were observed, and the tumor blood flow signal, resistance index (RI) and pulsability index (PI) under ultrasound were determined. Serum CA125 was detected by electrochemiluminescence, and VEGF and OPN levels were detected by enzyme-linked immunosorbent assay. The incidence of irregular lesion morphology, unclear boundary, uneven internal echo, microcalcification and side-acoustic images in OC group (OCG) was significantly higher than that in BCG (P<0.01). As for blood flow grading, most patients in the MTG were in grade Ⅱ and Ⅲ, while most patients in the BCG were in grade 0. Compared with BCG, the flow RI and PI in the OCG were significantly reduced (P<0.01). The levels of serum CA125, VEGF and OPN in OCG were significantly higher than those in BCG. The expression levels of serum CA125, VEGF and OPN in OC patients with clinical high stage (stage III and IV), poorly differentiated, ascites, recurrence and metastasis were significantly higher than those in patients with clinical low stage (stage I and II), well differentiated, no ascites and no recurrence and metastasis (P<0.05). With the disappearance of the tumor or the decrease of tumor load, the serum marker levels after treatment were significantly lower than that before treatment (P<0.05). The sensitivity and accuracy of the combined examination in the diagnosis of OC were obviously improved compared with the single and partial combined examinations (P<0.05). In conclusion, combined examination can significantly improve the sensitivity and accuracy of OC, which is conducive to early diagnosis and clinical intervention of OC.