Childhood acute lymphoblastic leukaemia (ALL) is suggested to result from a dysregulated immune response to infections in children with a preleukaemic state. Childcare in early life supposedly may protect against childhood ALL by facilitating sufficient exposure to infections to stimulate and ensure normal maturation of the immune system. We assessed the association between childcare attendance before age two years and risk of childhood ALL in a register-based cohort study, including all children aged 2-14 years born in Denmark during 1991-2014 with available childcare information recorded in the Danish Childcare Database (n=1,116,185). Cox regression was used to estimate hazard ratios (HRs) comparing children enrolled in childcare and children not enrolled before age two years. Further, we assessed the association according to age at enrolment, type of childcare facility and specific ALL subtypes. During 10,460,811 person-years of follow-up, 460 children developed ALL at ages 2-14 years. Of these, 57 (12.4%) never attended childcare before age two years compared with 10.6% in the total cohort. Compared with homecare, childcare attendance before age two years was associated with a statistically non-significantly, marginally decreased risk of childhood ALL with adjusted HR = 0.87 (95% confidence interval (CI): 0.65-1.16). Risk estimates did neither vary statistically significantly by age at enrolment nor by type of childcare facility and also not between childhood ALL subtypes, including frequently prenatally initiated ALL subtypes. Results from this large, nationwide register-based study provided no evidence that childcare attendance in the first years of life protects against childhood ALL.