To explore a 3.8-µm laser-induced damage and wound healing effect, we propose using optical coherence tomography (OCT) and a noninvasive monitoring-based in vivo evaluation method to quantitatively and qualitatively analyze the time-dependent biological effect of a 3.8-µm laser. The optical attenuation coefficient (OAC) is computed using a Fourier-domain algorithm. Three-dimensional (3-D) visualization of OCT images has been implemented to visualize the burnt spots. Furthermore, the burnt spots from the 3-D volumetric data was segmented and visualized, and the quantitative parameters of the burnt spots, such as the mean OACs, areas, and volumes, were computed. Then, OCT images and histological sections were analyzed to compare the structural changes. Within a certain radiation range, there is a linear relationship between radiation dose and temperature. Dermoscopic images, OCT images, and histological sections showed that, within a certain dose range, as the radiation doses increased, the cutaneous damage became more serious. One hour after laser radiation, the mean OACs increased and then decreased; the areas of burnt spots always increased and were 0.95 ± 0.07, 1.01 ± 0.06, 1.025 ± 0.07, 0.99 ± 0.07, 0.98 ± 0.07, 1.00 ± 0.07, 0.96 ± 0.05, and 0.98 ± 0.06 mm-1, respectively; the areas were 2.10 ± 0.63, 3.75 ± 1.85, 5.95 ± 1.62, 8.35 ± 0.88, 9.44 ± 1.28, 10.29 ± 0.49, 12.27 ± 0.96, and 13.127 ± 1.90 mm2; and the volumes were 1.54 ± 0.41, 2.86 ± 0.09, 3.73 ± 0.49, 4.14 ± 0.80, 7.21 ± 0.52, 6.77 ± 0.45, 8.36 ± 0.25, and 10.65 ± 0.51 mm3; and 21 days after laser radiation, the volumes were 0.67 ± 0.18, 1.64 ± 0.08, 1.87 ± 0.12, 2.57 ± 0.34, 3.43 ± 0.26, 3.64 ± 0.04, 3.84 ± 0.15, and 4.16 ± 0.53 mm3, respectively. We investigated the time-dependent biological effect of 3.8-µm laser-induced cutaneous damage and wound healing using the quantitative parameters of OCT imaging and noninvasive monitoring. The real-time temperature reflects the photothermal effect during laser radiation of mouse skin. OCT images of burnt spots were segmented to compute the mean OACs, burnt area, and quantitative volumes. This study has the potential for in vivo noninvasive and quantitative clinical evaluation in the future.