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Objective To investigate the effect of hospital working hours on outcomes of patients with acute ischemic stroke 3 months after receiving alteplase intravenous thrombolysis. Methods A retrospective analysis was performed on 254 individuals with acute ischemic stroke who received alteplase intravenous thrombolysis between January 2018 and December 2020 either during peak hospital working hours (08:00–17:59; Group A) or off-peak hours (18:00–07:59 the following day; Group B). Patients were also categorized according to which of four peak/off-peak-hour periods they received treatment in: Group 1 (08:00–11:59), Group 2 (12:00–17:59), Group 3 (18:00–21:59), Group 4 (22:00–07:59 the following day). Baseline data and 3-month prognosis were compared across groups. Logistic regression analysis was used to investigate the correlation between hospital working hours and 3-month prognosis. Results There were no significant differences in door-to-needle time, onset-to-needle time, 24-hour National Institutes of Health Stroke Scale (NIHSS) score, 7-day NIHSS score or Modified Rankin Score between Groups 1 to 4 or between Groups A and B. Whether treatment was administered during peak or off-peak hours did not significantly affect 3-month prognosis. Conclusion At this hospital, differences in the time at which stroke patients were treated were not associated with outcomes.