We discovered that several types of steroid hormones quench the fluorescence of quantum dots (QDs) at close proximity. Inspired by the finding, we developed a new type of biosensor for the sensitive detection of cortisol via direct fluorescence quenching of functionalized QD probes directly induced by the capture of target cortisol without additional reporter reagents. The detection selectivity was provided by cortisol-selective aptamers or anticortisol antibodies conjugated on the QD surfaces. With the magnetic nanoparticle labeling, the new sensing method enabled rapid cortisol sensing at physiologically relevant concentrations and yielded the detection limit of similar to 1 nM for aptamer-based sensors and similar to 100 pM for antibody-based sensors. We also evaluated the new detection method using saliva samples with an optimized sample preparation process under the assistance of magnetic manipulation. The result showed a satisfying recovery rate for spiked saliva tests. The facile sensing technology offers an appealing approach for the detection of steroid hormones in point-of-care settings.