Quantum dot (QD) imaging capability was investigated by the imaging depth at a near-infrared second optical window (SOW; 1000 to 1400 nm) using time-modulated pulsed laser excitations to control the effective fluence rate. Various media, such as liquid phantoms, tissues, and in vivo small animals, were used and the imaging depths were compared with our predicted values. The QD imaging depth under excitation of continuous laser was determined to be 10.3 mm for 2 wt% hemoglobin phantom medium and 5.85 mm for 1 wt% intralipid phantom, which were extended by more than two times on increasing the effective fluence rate to . Bovine liver and porcine skin tissues also showed similar enhancement in the contrast-to-noise ratio (CNR) values. A QD sample was inserted into the abdomen of a mouse. With a higher effective fluence rate, the CNR increased more than twofold and the QD sample became clearly visualized, which was completely undetectable under continuous excitation. Multiple acquisitions of QD images and averaging process pixel by pixel were performed to overcome the thermal noise issue of the detector in SOW, which yielded significant enhancement in the imaging capability, showing up to a 1.5 times increase in the CNR.