This study was aimed to analyze ocular biometric changes following cycloplegia in pediatric patients with strabismus and amblyopia. Cycloplegia is routinely used to measure refractive error accurately by paralyzing accommodation. However, effects on axial length (AL), anterior chamber depth (ACD), keratometry (Km), and white-to-white distance (WTW) are not well studied in this population. This retrospective study examined 797 patients (1566 eyes) undergoing cycloplegic refraction at a Samsung Kangbuk hospital pediatric ophthalmology clinic from 2010 to 2023. Ocular biometry was measured before and after instilling 1% cyclopentolate and 0.5% phenylephrine/0.5% tropicamide. Patients were categorized by strabismus diagnosis, age, refractive error and amblyopia status. Differences in AL, ACD, Km, WTW, and refractive error pre- and post-cycloplegia were analyzed using paired t tests. ACD (3.44 ± 0.33 vs 3.58 ± 0.29 mm, P < .05) and WTW (12.09 ± 0.42 vs 12.30 ± 0.60 mm, P < .05) increased significantly after cycloplegia in all groups except other strabismus subgroup (Cs) in both parameters and youngest subgroup (G1) in ACD. Refractive error demonstrated a hyperopic shift from −0.48 ± 3.00 D to −0.06 ± 3.32 D (P < .05) in overall and a myopic shift from −6.97 ± 4.27 to −8.10 ± 2.26 in high myopia (HM). Also, AL and Km did not change significantly. In conclusion, cycloplegia impacts ocular biometrics in children with strabismus and amblyopia, significantly increasing ACD and WTW. Refractive error shifts hyperopically in esotropia subgroup (ET) and myopically in high myopia subgroup (HM), eldest subgroup (G3) relating more to anterior segment changes than AL/Km. Understanding cycloplegic effects on biometry is important for optimizing refractive correction in these patients.