We have developed a digital light modulation system that utilizes a modified commercial projector equipped with a laser diode as a light source for quantitative measurements of in vivo tissue oxygenation in an unanesthetized zebrafish embryo via phase-based phosphorescence lifetime detection. The oxygen-sensitive phosphorescent probe (Oxyphor G4) was first inoculated into the bloodstream of 48 h post-fertilization (48 hpf) zebrafish embryos via the circulation valley to rapidly disperse probes throughout the embryo. The unanesthetized zebrafish embryo was introduced into the microfluidic device and immobilized on its lateral side by using a pneumatically actuated membrane. By controlling the illumination pattern on the digital micromirror device in the projector, the modulated excitation light can be spatially projected to illuminate arbitrarily-shaped regions of tissue of interest for in vivo oxygen measurements. We have successfully measured in vivo oxygen changes in the cardiac region and cardinal vein of a 48 hpf zebrafish embryo that experience hypoxia and subsequent normoxic conditions. Our proposed platform provides the potential for the real-time investigation of oxygen distribution in tissue microvasculature that relates to physiological stimulation and diseases in a developing organism.