Although early reperfusion therapy is effective for acute ischemic stroke, limited therapeutic time-window resulted in only 10% of patients receiving reperfusion therapy. A fast and reliable stroke detection method is desired so that patients can receive early reperfusion therapy. It has been reported that ischemic stroke affects heart rate variability (HRV), which reflects activities of the autonomic nervous function. Thus, ischemic stroke may be detected at an acute stage through monitoring HRV. This paper proposes an HRV-based ischemic stroke detection algorithm by using multivariate statistical process control (MSPC), which is a well-known anomaly detection algorithm. As a feasibility study before collecting a large amount of clinical data from human patients, this paper used the middle cerebral artery occlusion (MCAO) model in rats for collecting HRV data shortly after ischemic stroke onsets. The 11 MCAO-operated rats and 11 sham-operated rats were prepared, and HRV data of three sham-operated rats were used for model construction. The data on the other 19 rats were used for its validation. The experimental result showed that sensitivity and specificity of the proposed algorithm were 82% and 75%, respectively. Thus, the present work shows the possibility of realizing an HRV-based ischemic stroke detection system for human patients.