A novel attempt was made to develop a disposable multifunctional sensor for analysis of vaccinia virus (VACV), a promising oncolytic agent that can replicate in and kill tumor cells. Briefly, we developed aptamers specific to VACV that were negatively selected against human serum as well as human and mouse blood to be further utilized for viral analysis directly in serum and blood. In addition, the aptamers were negatively selected against heat-inactivated VACV to enable them to distinguish between viable and nonviable virus particles. The selected aptamers were integrated onto an electrochemical aptasensor to perform multiple functions, including quantification of VACV, viability assessment of the virus, and estimation of the binding affinity between the virus and the developed aptamers. The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VACV-specific aptamer onto a gold nanoparticles modified screen-printed carbon electrode (GNPs-SPCE). Square wave voltammetry was employed to quantify VACV in serum and blood within the range of 150-900 PFU, with a detection limit of 60 PFU in 30 μL. According to the electrochemical affinity measurements, three virus specific aptamer clones, V-2, V-5, and V-9 exhibited the highest affinity to VACV. Furthermore, flow cytometry was employed to estimate the dissociation constants of the clones which were found to be 26.3, 40.9, and 24.7 nM, respectively. Finally, the developed aptasensor was able to distinguish between the intact virus and the heat-inactivated virus thanks to the tailored selectivity of the aptamers that was achieved via negative selection.