AIM: To evaluate the effect of different frequencies 0.01% atropine eye drops on the ocular surface in adolescent myopia control. METHODS: A total of 84 adolescent patients with myopia were recruited for this study. They were randomly assigned to receive 0.01% atropine eye drops at different frequencies (morning group, evening group, morning and evening group). During the treatment period, ocular and systemic adverse reactions were observed, spherical equivalent refraction (SER) and axial length (AX) were examined. Additionally, ocular surface disease index (OSDI) questionnaire was obtained, tear meniscus height (TMH) ,non-invasive first tear film break-up time (NifBUT), non-invasive average tear film break-up time (NiaBUT), and meibomian gland atrophy score (MaS) were measured by Keratograph 5M. RESULTS: During the 6-month treatment with 0.01% atropine eye drops, all adverse reaction  symptoms were relatively mild and infrequent. Only one case of systemic reaction was reported, with temporary xerostomia. Six patients reported discomfort such as ocular itching and dryness. The ocular reactions of photophobia and blurred near vision were slightly more common in the morning and combined morning-evening groups compared to the evening group, but there were no significant differences among the three groups (P>0.05). Compared to baseline, there were significant differences in SER and AX among the three groups after six months of treatment (P<0.05). Compared to baseline, no significant differences were observed in the Morning group after six months of treatment in terms of OSDI score (P=0.656), TMH value (P=0.362), NifBUT (P=0.510), NiaBUT (P=0.700), and MaS (P=0.846). Similarly, no significant changes were found in the Evening group regarding OSDI score (P=0.429), TMH value (P=0.462), NifBUT (P=0.462), NiaBUT (P=0.598), and MaS (P=0.351). Additionally, in the Morning and evening group, no significant changes were detected in OSDI score (P=0.457), TMH value (P=0.748), NifBUT (P=0.197), NiaBUT (P=0.070), and MaS (P=0.802) after six months of treatment. CONCLUSION: After six months of using 0.01% atropine eye drops with different frequencies in adolescent myopia control, there was no significant impact on the ocular surface.