This study presents a selective impedimetric biosensor for the detection of A29, a target protein of the monkeypox virus (MPXV). The biosensor operates based on the interaction between A29, an internal membrane protein of MPXV, and the heparan sulfate receptor. The interaction between A29 and heparan sulfate (HS) was monitored using electrochemical impedance spectroscopy (EIS) after immobilizing HS on a gold screen-printed electrode (AuSPE). The biosensor exhibited a linear detection range of 2.0–50 ng mL⁻¹ with a detection limit of 2.08 ng mL⁻¹ and a quantification limit of 6.28 ng mL⁻¹. A relative standard deviation of 2.82% was observed for 25 ng mL⁻¹. The biosensor was also tested with real serum samples, showing good recovery values. The developed biosensor offers a rapid, effective, and economical method for MPXV detection, with high specificity due to the A29-HS interaction. It is suitable for point-of-care (POC) applications and could be used as a diagnostic tool for future MPXV outbreaks. The biosensor was compared with CRISPR methods, showing better practicality and speed. Interference studies with potential interferents (A27, KMP11, H1N1) showed high recovery rates, indicating the biosensor's robustness. The study highlights the potential of the developed biosensor for MPXV detection in clinical settings.This study presents a selective impedimetric biosensor for the detection of A29, a target protein of the monkeypox virus (MPXV). The biosensor operates based on the interaction between A29, an internal membrane protein of MPXV, and the heparan sulfate receptor. The interaction between A29 and heparan sulfate (HS) was monitored using electrochemical impedance spectroscopy (EIS) after immobilizing HS on a gold screen-printed electrode (AuSPE). The biosensor exhibited a linear detection range of 2.0–50 ng mL⁻¹ with a detection limit of 2.08 ng mL⁻¹ and a quantification limit of 6.28 ng mL⁻¹. A relative standard deviation of 2.82% was observed for 25 ng mL⁻¹. The biosensor was also tested with real serum samples, showing good recovery values. The developed biosensor offers a rapid, effective, and economical method for MPXV detection, with high specificity due to the A29-HS interaction. It is suitable for point-of-care (POC) applications and could be used as a diagnostic tool for future MPXV outbreaks. The biosensor was compared with CRISPR methods, showing better practicality and speed. Interference studies with potential interferents (A27, KMP11, H1N1) showed high recovery rates, indicating the biosensor's robustness. The study highlights the potential of the developed biosensor for MPXV detection in clinical settings.