Low back pain (LBP) is a highly prevalent disease. Among the various causes of LBP, one of the most frequent is myofascial pain syndrome (MPS) which affects the spinal stabilizer muscles. The aims of this study were to compare the differences in muscular electrical activity and biomechanical properties between the painful and non-painful sides in patients with unilateral MPS and to verify the feasibility of surface electromyography (sEMG) and MyotonPRO for assisting in MPS assessment. Forty patients with unilateral lumbar MPS were recruited via the Department of Rehabilitation Medicine Center of West China Hospital Sichuan University from October 2022 to October 2023. The electrical properties of the bilateral erector spinae muscles were characterized by sEMG signals during a trunk extension task. The following four time-domain features of sEMG were extracted: root mean square (RMS), mean absolute value (MAV), integrated EMG (iEMG), and waveform length (WL). And two frequency domain features were extracted: the median frequency (MDF) and mean power frequency (MPF). The mechanical properties of the muscles were assessed by MyotonPRO at rest. The following biomechanical parameters were acquired: oscillation frequency [Hz], dynamic stiffness [N/m], logarithmic decrement, relaxation time [ms], and Creep. The visual analog scale (VAS) was used to evaluate the pain severity, and the Oswestry Disability Index (ODI) was used to evaluate the severity of disability and disruption to lifestyle activities caused by LBP pain. The outcome measures were obtained prior to the Platelet-rich plasma (PRP) treatment and repeated two weeks after treatment. (1) Prior to the PRP treatment, all sEMG time-domain features on the painful side were significantly higher than those on the non-painful side (RMS, p < 0.001; MAV, p < 0.001; iEMG, p < 0.001; WL, p = 0.001). However, there was no significant difference in the sEMG frequency-domain features (MPF, p = 0.478; MDF, p = 0.758). On the mechanical side, there were significant differences in oscillation frequency (p = 0.041) and logarithmic decrement (p = 0.022) between the painful side and non-painful side, but no significant differences in dynamic stiffness, relaxation time, and creep (both p > 0.05). (2) Two weeks after the PRP treatment, statistically significant decreases were observed in both post-treatment VAS (p < 0.001) and ODI scales (p < 0.001), indicating the PRP treatment clinically significantly reduced the level of. MPS. This change coincided with all sEMG time-domain features, in which the values at the painful side decreased significantly (RMS, p = 0.001; MAV, p = 0.001; iEMG, p = 0.001; WL, p = 0.001). However, no significant difference in the sEMG frequency-domain features (MPF, p = 0.620; MDF, p = 0.850) was found. On the mechanical side, only logarithmic decrement on the painful side increased significantly (p < 0.001). Our combined MyotonPRO and sEMG results indicated that MPS likely leads to increased muscle tone and decreased muscle elasticity, manifested by abnormal time-domain features of sEMG and biomechanical properties. The changes in these objective measurements were agreed with the changes in subjective outcome measures of pain and function currently assessed in the patients with MPS. A single PRP treatment may alleviate muscle dysfunction caused by MPS. These preliminary results demonstrated the potential feasibility of using sEMG and MyotonPRO as tools for assessing the neuromuscular function of MPS.