Introduction: Hand, foot, and mouth disease (HFMD) has been an important public health concern worldwide, especially in the Asia-Pacific region. Unfortunately, the effect of current measures on preventing and controlling HFMD may be limited. Isolation of infectious sources is reported as an important way to prevent and control this disease. The isolation period is determined on the basis of duration of viral shedding in patients with HFMD. However, the results of previous researches on duration of viral shedding remain controversial. Here, we present a protocol of a systematic review and single-arm meta-analysis for assessing the duration of viral shedding in patients with HFMD induced by Enterovirus 71 (EV71), Coxsackievirus A16 (CA16), or Coxsackievirus A6 (CA6). Methods and analysis: A comprehensive literature search will be performed in PubMed, EMBASE, the Cochrane library, Chinese National Knowledge Infrastructure (CNKI), Chinese Biomedical Literature Database (CBM), and Wanfang Database, covering the period from inception to May 1, 2019. Point estimate of positive rate with corresponding 95% confidence intervals (CIs) of EV71, CA16, or CA6 in HFMD patients' fecal or throat samples will be carried out using STATA 14.0. Subgroup analyses will be performed for mild cases, severe cases, and close contacts. Sensitive analysis will also be performed to evaluate the influences of individual studies on the final effect by exclusion of a few articles of poor quality. We will assess the risk of bias for the final studies included in our meta-analysis using previously available tools and the modified risk of bias tool. Results: The results of this systematic review and meta-analysis will be published in a peer-reviewed journal. Conclusion: To the best of our knowledge, this paper will be the first systematic review and meta-analysis for assessing the duration of viral shedding in patients with HFMD induced by EV71, CA16, or CA6. The conclusions drawn from this review will provide the scientific basis to formulate the isolation period of HFMD. Ethics and dissemination: Ethical review is not required as this article is for a systematic review since there is no direct involvement of patients in the whole process. We will publish the results of this systematic review and meta-analysis of single-arm studies in a peer-reviewed journal. Registration number: Prospero CRD42020139999.