Purpose: To evaluate the prevalence of amblyopia and associated biometric factors in Danish children. Methods: Determination of best-corrected visual acuity (BCVA) using ETDRS charts, non-cycloplegic subjective refractioning guided by automated refractometry, axial length and corneal curvature, fundus photography and optical coherence tomography (OCT) in 1335 children from the population-based Copenhagen Child Cohort 2000 (CCC2000) Eye Study. Birth data were obtained from the Danish Medical Birth Registry. Results: The mean (±SD) age of children was 11.7 (±0.4) years, and 47% were boys. Amblyopia prevalence was 1.5 (95% CL 0.8–2.2) %. Unilateral amblyopic eyes [BCVA < 80 ETDRS letters (0.8 snellen) and ≥2 lines difference between the eyes] was 0.6 (95% CL 0.3–1.0) mm shorter, 1.34 (95% CL 0.30–2.37) D more hyperopic and had 0.79 (95% CL 0.14–1.44) D more astigmatism compared with fellow eyes. Compared with the right eyes of the non-amblyopic children, unilateral amblyopic eyes were 1.0 (95% CL 0.5–1.6) mm shorter, 2.48 (95% CL 1.11–3.86) D more hyperopic, 1.09 (95% CL 0.43–1.75) D more astigmatic and had a 47 (95% CL 13–81) μm thicker subfoveal choroid. Conclusion: Amblyopia was found in 1.5% of Danish children born 22 years after the inception of the nationwide preschool visual screening programme. Amblyopia was associated with anisometropia, astigmatism, a thicker subfoveal choroid and a history of childhood strabismus.