Ocular pain is a common complication following photorefractive keratectomy (PRK). The level of patient satisfaction with current pain control strategies is not high. This study aims to assess the efficacy and safety of a novel regimen of preservative-free oxybuprocaine hydrochloride 0.4% unit-dose eye drops for post-PRK pain control. In a contralateral eye study, 144 eyes of 72 patients who underwent bilateral transepithelial PRK (TransPRK) were stratified into experimental and control groups. The experimental group received preservative-free oxybuprocaine hydrochloride 0.4% unit-dose eye drops five times daily postoperatively until complete epithelial healing, while the control group received sodium hyaluronate 0.2% instead. The main outcome measures were pain scores assessed by the verbal rating scale and visual analogue scale (VRS, VAS), the corneal epithelial defect (CED) area, epithelial healing duration evaluated by slit-lamp biomicroscopy and anterior segment optical coherence tomography (AS-OCT), and endothelial cell density (ECD) measured before and 1 month after surgery. Pain scores assessed by VRS and VAS were significantly lower in the experimental group 8h after surgery, and 1, 2, and 3days postoperatively (P < 0.001). The mean CED area showed no significant differences between the two groups at different follow-ups (P value > 0.05). The corneal epithelial healing had a mean duration of 3.32 ± 0.47 days in both studied groups and was parallel in both eyes of each patient. In each group, 49 eyes (68%) and 72 eyes (100%) had a fully epithelialized surface on the third and fourth postoperative days, respectively. No significant changes were observed in the mean ECD 1 month following surgery in both groups (P value > 0.05). Preservative-free oxybuprocaine hydrochloride 0.4% unit-dose eye drops are effective and safe in controlling early postoperative pain following TransPRK. The availability of the single-dose unit preparation can overcome the problem of topical anesthetic abuse. ClinicalTrials.gov Identifier: NCT05733741.