Cancer is one of the most tormenting global health burdens reporting high mortality and morbidity worldwide. Matrix metalloproteinase 2 (MMP-2) protein has elevated expression for most types of cancers, including prostate and breast cancer. Therefore, accurate and specific detection of MMP-2 biomarker is crucial for screening, treatment, and prognosis of related cancer. In this work, we have proposed a label-free electrochemical biosensor for the detection of MMP-2 protein. This biosensor was fabricated using hydrothermally synthesized vanadium disulfide (VS2) nanosheets with monoclonal anti-MMP2 antibodies biofunctionalized using a suitable linker. The VS2 nanomaterials were synthesized hydrothermally at different reaction temperatures (140 °C, 160 °C, 180 °C and 200 °C) generating different morphologies from a 3D bulk cubic structure at 140 °C to 2D nanosheets at 200 °C. Owing to the advantages of 2D VS2 nanosheets with high surface-to-volume ratio, excellent electrochemical response and high antibody loading possibility, it was selected for fabricating an MMP-2 specific biosensor. The antibody-antigen binding event is analyzed by recording electrochemical impedance spectroscopy signals for different target MMP-2 protein concentrations. The sensitivity and lower limit of detection were 7.272 (ΔR/R)(ng ml)−1 cm−2 and 0.138 fg ml−1, respectively in 10 mM phosphate buffer saline for this proposed sensor. Further, interference studies were also performed which demonstrates the sensor to be highly selective against non-specific target proteins. This 2D VS2 nanosheet-based electrochemical biosensor is a sensitive, cost-effective, accurate, and selective solution for cancer diagnosis. © 2023 IOP Publishing Ltd.