Objective To evaluate the value of serum β-human chorionic gonadotropin (β-hCG) for predicting the outcome of ectopic pregnancy treated with single-dose intramuscular injection of methotrexate (MTX). Methods The clinical data of 96 ectopic pregnancy patients treated with MTX intramuscular injection in Changhai Hospital of Naval Medical University (Second Military Medical University) from Jan. 1, 2010 to Dec. 31, 2018 were retrospectively analyzed. All patients received MTX single-dose (50 mg/m²) intramuscular injection, and the serum β-hCG level was detected regularly. After receiving MTX single-dose intramuscular injection, the patients whose serum β-hCG levels were reduced to the normal twice, and who did not receive the second MTX treatment or surgical treatment were considered as successful. According to the treatment outcome, the patients were divided into treatment success group and failure group. The serum β-hCG levels of the two groups were compared before treatment. According to the pre-treatment serum β-hCG levels, they were divided into <1 000, 1 000-1 999, 2 000-2 999 and ≥ 3 000 mIU/mL groups, and the success rates of MTX single-dose injection were compared among the groups. The prognostic value of serum β-hCG was analyzed at different time points after treatment. Results Of the 96 ectopic pregnancy patients, 57 (59.4%) cases were treated successfully with MTX single-dose injection, and 39 (40.6%) cases were failed. The pre-treatment serum β-hCG levels were 343.00 (212.50, 720.00) and 716.00 (341.00, 1 619.00) mIU/mL in the treatment success group and failure group, respectively, with significant difference (P=0.007). With the increase of pre-treatment serum β-hCG level, the success rate of MTX single-dose injection decreased gradually; however, there were no significant differences in the success rates among the subgroups with different levels of β-hCG when the pre-treatment β-hCG level was lower than 3 000 mIU/mL (P>0.05). The area under curve (AUC) of serum β-hCG 4 days after treatment in predicting the success of MTX single-dose injection was 0.80 (95% confidence interval[CI] 0.70-0.88, P<0.001), the cut-off value was 650 mIU/mL, the sensitivity was 91.2%, and the specificity was 66.7%; the AUC of serum β-hCG 7 days after treatment in predicting the success of MTX single-dose injection was 0.71 (95% CI 0.58-0.82, P<0.001), the cut-off value was 103 mIU/mL, the sensitivity was 42.9%, and the specificity was 100.0%. The success rate in the group with serum β-hCG ≤ 650 mIU/mL 4 days after treatment was significantly higher than that in the group with serum β-hCG >650 mIU/mL (P<0.001), while the success rates were similar in the groups with serum β-hCG ≤ 103 mIU/mL and >103 mIU/mL 7 days after treatment (P=0.146). Conclusion For ectopic pregnancy patients receiving a single-dose injection of MTX, the initial β-hCG levels are lower in patients with successful treatment. Serum β-hCG ≤ 650 mIU/mL on day 4 after treatment could be a predictor of treatment success.