Mefloquine (MQ) is a quinoline based antimalarial drug, which is potent against multiple drug‐resistant Plasmodium falciparum. It is widely prescribed for the prophylactic treatment of malaria. Due to extensive usage of MQ, constant monitoring of the drug level in human body is of paramount importancein order to ensure that optimum drug exposure is achieved. The present work describes a gold nanourchins (AuNUs) based electrochemical sensor for the determination of MQ.AuNUs were synthesized via seed‐mediated method and characterized using ultraviolet‐visible spectroscopy, energy‐dispersive X‐ray spectroscopy, field emission scanning electron microscopy, zeta‐sizer and electrochemical techniques (electrochemical impedance spectroscopy and cyclic voltammetry). Fabrication of the sensor was done by drop‐coating the synthesized AuNUs onto a glassy carbon electrode. The fabricated sensor exhibited enhanced voltammetric response, which was attributed to the excellent conductivity and high surface area of AuNUs. Under optimum square wave voltammetric conditions, the sensor displayed two linear response ranges (from 2.0×10−9 to 1.0×10−6 M and from 1.0×10−6 to 1.0×10−3 M) with a detection limit of 1.4 nM. The electrode demonstrated good reproducibility, stability and selectivity over common interferents. The utility of the sensor was successfully assessed for quantification of the drug in pharmaceutical preparation and spiked human urine sample. Thus, the present study demonstrates a promising approach for determination of MQ with practical utility in quality control and clinical analysis.