Background Hemiplegic shoulder pain (HSP) is a common symptom for post-stroke patients, which has a severely adverse impact on their rehabilitation outcomes. However, the cause of HSP has not been clearly identified due to its complicated multifactorial etiologies. As possible causes of HSP, the abnormality of both muscular electrical activity and blood perfusion remains lack of investigations. Objective This study aimed to analyze the alteration of muscular electrical activity and blood perfusion of upper extremity in patients with HSP by using surface electromyography (sEMG) and laser speckle contrast imaging (LSCI) measurement techniques, which may provide some insight into the etiology of HSP. Methods In this observational and cross-sectional study, three groups of participants were recruited. They were hemiplegic patients with shoulder pain (HSP group), hemiplegic patients without shoulder pain (HNSP group), and healthy participants (Healthy group). The sEMG data and blood perfusion data were collected from all the subjects and used to compute three different physiological measures, the root-mean-square (RMS) and median-frequency (MDF) parameters of sEMG recordings, and the perfusion unit (PU) parameter of blood perfusion imaging. Results The RMS parameter of sEMG showed significant difference (p < 0.05) in the affected side between HSP, HNSP, and Healthy groups. The MDF parameter of sEMG and PU parameter of blood perfusion showed no significant difference in both sides among the three groups (p > 0.05). The RMS parameter of sEMG showed a statistically significant correlation with the pain intensity (r = -0.691, p =0.012). Conclusion This study indicated that the muscular electrical activity of upper extremity had a correlation with the presence of HSP, and the blood perfusion seemed to be no such correlation. The findings of the study suggested an alternative way to explore the mechanism and treatment of HSP.