Early diagnosis of wound infections that aid in the assessment of the stage of wound infection is gaining interest for development of a new biomarker. In present research, a simple and portable 3D origami-fluidic paper-based electrochemical sensor is proposed for the detection of myeloperoxidase (MPO) activity in wound exudate samples. The flow can be manipulated by an origami pattern of the paper allowing the electrochemical reaction to occur on the electrode surface. Glucose oxidase (GOx) was used for producing hydrogen peroxide (H2O2) and MPO antibodies were immobilized to capture the MPO to enhance the specificity. Prussian blue/MXene composite modified on the electrode act as a nanozyme for catalyzing H2O2. Then, data is wirelessly transferred to a smartphone using near-field communication (NFC) technology. Under the optimal conditions, our proposed device can detect MPO activity in ranges 0.5–5 U/ml (R2 =0.9697), demonstrating a strong correlation with a colorimetric MPO activity kit when tested with real wound fluid samples (n = 6, R2 = 0.9198). Notably, our method can differentiate between non-infected and infected wounds (p-value < 0.05) by analyzing MPO activity extracted from used wound-dressing gauzes (n = 10). Therefore, our device offers a rapid portable device for MPO activity that is promising for resource-limited settings.