Rapid and accurate detection of glucose is of great significance for diabetic management. Highly sensitive glucose sensors promise to achieve noninvasive detection technology, enabling more convenient and efficient means for large-scale screening and long-term dynamic monitoring of diabetes patients. In this work, we demonstrate a sensitive glucose electrochemical biosensor through the synergetic labelling strategy utilizing PbS colloidal quantum dots (CQDs) and Au nanospheres (AuNSs). The PbS CQDs/AuNSs/glucose oxidase (GOx) mixture could be stably immobilized on the carbon electrode surface via the one-step dip-coating method. The electrochemical biosensor employing PbS CQDs/AuNSs/GOx-modified electrode integrates the functions of specific molecule recognition, signal transduction as well as signal amplification. The sensor is capable of transducing the glucose enzyme-catalyzed reaction into significant current signals, exhibiting a good linear response in the glucose concentration range of 0.1 μM–10 mM with the limit of detection being 1.432 nM.