This study presents a selective impedimetric biosensor for the detection of A29, a protein specific to the monkeypox virus (MPXV). The biosensor's working principle is based on the interaction between A29 and heparan sulfate (HS), which is immobilized on a gold screen-printed electrode (AuSPE). Electrochemical impedance spectroscopy (EIS) was used to monitor the interaction. After optimizing experimental parameters, the biosensor exhibited a linear detection range of 2.0 to 50 ng mL⁻¹, with a detection limit of 2.08 ng mL⁻¹ and a quantification limit of 6.28 ng mL⁻¹. The relative standard deviation for 25 ng mL⁻¹ A29 was 2.82%. The biosensor was also tested with real serum samples from healthy individuals, showing good recovery values. The developed biosensor is a promising tool for rapid, effective, and economical detection of MPXV, particularly in the context of the global public health emergency caused by the monkeypox virus.This study presents a selective impedimetric biosensor for the detection of A29, a protein specific to the monkeypox virus (MPXV). The biosensor's working principle is based on the interaction between A29 and heparan sulfate (HS), which is immobilized on a gold screen-printed electrode (AuSPE). Electrochemical impedance spectroscopy (EIS) was used to monitor the interaction. After optimizing experimental parameters, the biosensor exhibited a linear detection range of 2.0 to 50 ng mL⁻¹, with a detection limit of 2.08 ng mL⁻¹ and a quantification limit of 6.28 ng mL⁻¹. The relative standard deviation for 25 ng mL⁻¹ A29 was 2.82%. The biosensor was also tested with real serum samples from healthy individuals, showing good recovery values. The developed biosensor is a promising tool for rapid, effective, and economical detection of MPXV, particularly in the context of the global public health emergency caused by the monkeypox virus.