Laser treatment is the most effective therapy in dermatology for vascular skin disorders, such as port-wine stains (PWS). Changes in heat-induced absorbance in blood must be determined for accurate numerical simulation and implementation of multi-pulse laser therapy for treatment of PWS. Thermally induced absorbance changes in hemoglobin in blood were compared in vitro between slow water bath heating and fast heating irradiated by using sub-millisecond Nd:YAG laser. Blood composition at different temperatures was calculated by comparing blood absorption spectra with those of pure HbO, Hb, and metHb at room temperature. Blood absorbance to heat energy were categorized into three stages distinguished by metHb and coagulation points, which are the validity and security thresholds of the optimized therapy, respectively. Rapid laser heating can distinctively enhance blood absorbance by photochemically induced strong instability compared with slow heating at a constant temperature. Slow heating facilitates metHb point at 70 °C and coagulation point at 75 °C as the temperature of the water bath increases. However, the temperature at which metHb or coagulation point shifts to higher than 10 °C when pulses and fluence in laser irradiation change. Laser fluence less than 20 J/cm and more than 50 J/cm is unsuitable for laser treatment because of its low probability to coagulate vascular hyperplasia and high probability to damage normal tissues adjacent to target lesions, respectively. Few bubbles formed after mediate fluence is beneficial to minimize adverse side-effects. Considering blood absorbance, temperature evolution, and bubble formation, we recommend 30-40 J/cm and 2-4 Hz frequency as the optimal laser parameters in sub-millisecond Nd:YAG laser.