Background and purpose Neutrophils, a pivotal immune responder to ischaemic brain insult, have been involved in neuroplasticity and increase after stroke. Recombinant tissue plasminogen activator (r‐tPA), a promising treatment improving neuroplasticity, promotes neutrophil degranulation. However, the dynamic profile of neutrophils after r‐tPA treatment and their effect on neurological recovery after stroke are not well studied. Methods Cell counts of neutrophils, lymphocytes and their ratio (NLR) were measured on admission and 24 h after r‐tPA infusion in 372 consecutively recruited acute ischaemic stroke patients (mean age 64 years). Death or major disability at 3 months after stroke was diagnosed based on the modified Rankin Scale (mRS ≥ 3) obtained by neurologists who were blinded to any hospital records. The longitudinal associations of percentage increase in neutrophils, lymphocytes and the NLR with death or major disability were examined by logistic regression adjusting for covariates including neurological deficits at baseline. Results Neutrophils exhibited a steeper increase after r‐tPA infusion in patients with death or major disability than in those without (P < 0.001). A 10% increase in neutrophils after r‐tPA infusion was associated with an 83% increased risk for death or major disability within 3 months after stroke onset odds ratio (OR) 1.99, P = 0.009. Increased neutrophils at 24 h after r‐tPA (OR 6.30, P < 0.001 after log transformation) but not on admission significantly predicted increased risks for death or major disability within 3 months after stroke onset. A similar phenomenon was also observed for the NLR. Conclusions A dynamic increase in neutrophils after stroke significantly predicts 3‐month death or major disability in acute ischaemic stroke patients receiving r‐tPA treatment.