In the epoch of bioinformatics, pivotal biomedical scrutiny and clinical diagnosis hinge upon the unfolding of highly efficacious biosensors for intricate and targeted identification of specific biomolecules. In pursuit of developing robust biosensors endowed with superior sensitivity, precise selectivity, rapid performance, and operational simplicity, semiconductor QDs have been acknowledged as pivotal and advantageous entities. In this review, we present a comprehensive analysis of the latest unfolding within the domain of QDs used in fluorescent biosensors for the detection of diverse biomolecular entities, encompassing proteins, nucleic acids, and a range of small molecules, with an emphasis on the synthesis methodologies of QDs employed and mechanism behind sensing. Additionally, this review delves into several pivotal facets of QD-based fluorescent biosensors in detail, such as surface functionalization methodologies aimed at enhancing biocompatibility and improving target specificity. The challenges and future perspectives of QD-based fluorescent biosensors are also considered, emphasizing the necessity of ongoing multidisciplinary research to realize their full potential in enhancing personalized medicine and biomedical diagnostics.