BACKGROUND: As the priority drug for treating acute ischemic stroke (AIS), alteplase is a thrombolytic drug with strong fibrin specificity. It can obviously treat AIS with high safety. However, the validity of its time window is controversial. This study focus on the efficacy and safety of intravenous thrombolysis with alteplase for treating AIS at different time windows. METHODS: Retrieval of English database (PubMed, Embase, Web of Science, the Cochrane Library) and Chinese database was conducted (China National Knowledge Infrastructure, WAN FANG, VIP, China Biology Medicine disc) by computers. From the establishment of the database to October 2020, a retrospective study and case-control study on intravenous thrombolysis at different time windows for treating AIS were conducted. Two researchers independently conducted data extraction and quality evaluation of literature on the included studies, and RevMan5.3 was used for Meta-analysis on the included literature. RESULTS: This study aims to evaluate the efficacy and safety of intravenous thrombolysis with alteplase at different time windows for treating AIS by National Institutes of Health Stroke Scale score, modified Rankin Scale rating scale, spontaneous intracerebral hemorrhage incidence rate, All-cause mortality, and so on. CONCLUSIONS: This study will provide an evidence-based basis for the clinical efficacy of alteplase for treating AIS by thrombolytic therapy at different time windows. ETHICS AND DISSEMINATION: Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval was not required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605 / OSF.IO / K7PHB. Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.