Aim: A tubal ectopic pregnancy (EP) in the first trimester remains a major life-threatening complication for the mother. We aim to determine whether serum reproductive hormones may be clinically useful in the early identification of a tubal EP. Methods: A total of 109 age-matched patients with a serum b-human chorionic gonadotropin (b-hCG) concentration <2000 IU/L were enrolled, including 68 patients with a tubal EP, 22 with a viable intrauterine pregnancy (vIUP) and 19 with a non-viable intrauterine pregnancy (nIUP). Serum was collected during the first trimester of pregnancy and assayed for b-hCG, follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (TE), progesterone (P), estradiol (E2) and prolactin (PRL) by using automated electrochemiluminescence immunoassays. Results: Patients with an EP had significantly lower levels of daily b-hCG variation (Db-hCG/day), P and E2, and significantly higher levels of LH and FSH than IUP patients (P < 0.05). As an EP diagnostic marker, progesterone demonstrated a sensitivity of 100% at the cutoff of 86.01 nmol/L. The combination of E2 with Db-hCG/day reached a specificity of 100% for EP evaluation. To identify non-viable pregnancies (including EPs and nIUPs), progesterone demonstrated a sensitivity of 95.40% and a specificity of 90.91% at the cutoff of 63.2 nmol/L; the diagnostic power of the receiver operating curve was 0.9702. Conclusions: A combination of Db-hCG/day, P and E2 may help distinguish EPs and nIUPs from vIUPs, facilitating earlier diagnosis and the timely implementation of medical treatment to prevent tubal rupture.