The preservation of ovarian reserve during laparoendoscopic single-site (LESS) ovarian cystectomy is crucial for reproductive-age women. This study was a single-blinded, single-center, and randomized controlled trial to evaluate the effect of hemostatic agents on the preservation of ovarian reserve and hemostasis during LESS ovarian cystectomy. Patients with unilateral ovarian cyst were randomized to the hemostatic agent and coagulation groups according to the hemostasis method. Afterwards, the patients underwent LESS ovarian cystectomy, and hemostasis was performed after ovarian cyst excision according to the assigned hemostasis method. If hemostasis was not completed within 10 min. After discharge, the patients were followed until 3 months after surgery. We compared the hemoglobin, anti-Müllerian hormone (AMH) levels, and ovarian volumes before surgery, and 2 days, 1 week, and 3 months after surgery (3 M-POST), and the decline ratio between the two groups. The decline ratio of serum AMH levels was greater at 3 M-POST in the coagulation than in the hemostatic agent group (median intention-to-treat [ITT], − 36.7 vs. − 13.3%; per-protocol [PP], − 36.8 vs. − 13.3%; P < 0.05). Notably, the difference of the decline ratio of serum AMH levels was only shown in endometriosis patients (median; ITT, − 50.7 vs. − 14.4%; PP, − 50.7% vs. − 14.4%; P < 0.05), while there was no difference in non-endometriosis patients. In conclusion, Hemostatic agents may be non-inferior to bipolar coagulation for preserving ovarian reserve and hemostasis during LESS ovarian cystectomy, in particular, for endometriosis patients. (Trial registry: ClinicalTrials.gov Identifier NCT03374397).