Excess and unmonitored use of antibiotics across globe is rapidly leading to antibiotics resistance. Thus, early-stage detection of antibiotics resistance has become necessary to monitor and control their use to reduce the side effects. Amoxicillin (Amoxi) is a type of beta-lactam antibiotic that is often used to treat bacterial infections. Herein, L-cysteine capped vanadium disulphide quantum dots (L-CYST-VS2QDs) were synthesized via hydrothermal method and used to construct an electrochemical immunosensor for Amoxi detection. To prepare Ab-Amoxi/L-CYST-VS2QDs/ indium tin oxide coated glass (ITO) immunoelectrode, monoclonal antibodies against Amoxi were immobilized on the L-CYST-VS2QDs/ITO followed by blocking uncovered sites with BSA. This BSA/Ab-Amoxi/L-CYST-VS2/ITO bioelectrode was revealed selective and specific interaction towards Amoxi among various other antibiotics. The immunoelectrode showed linearity within 1 pM to 100 µM range, with sensitivity of 4.827 µA nM−1 cm2, and detection limit of 0.5 pM, with better results than chitosan–TGA-VS2QDs based immunosensor. The applicability in real sample testing was proved by monitoring the response in spiked fish samples, which yielded positive outcomes.