<p>In cancer tissue diagnostic studies of clinical medicine, cell morphology is a key indicator for assessing the behavior of cellular physiological activities. Currently, the method of using thermotherapy as an adjunct to cancer treatment has gradually become a trend. Assessing the morphological characteristics of wound tissue cells during the hyperthermia process is particularly important for providing feedback on the therapeutic efficacy of hyperthermia-assisted treatment. Among the existing cell observation techniques, optical bright-field microscopy can only perform static observations of cells from a two-dimensional planar perspective. However, fluorescence microscopy suffers from issues such as phototoxicity and low temporal resolution. To address the aforementioned issues, this study introduces a quantitative analysis method based on digital holography to overcome these limitations. According to the mechanism of hyperthermia in cancer treatment, cancer cells exhibit morphological changes when exposed to elevated temperatures. Digital holography technology can effectively utilize light refractive indexes and phase differences to quantify the thickness and volume of cells. This study systematically evaluated the morphological changes in HeLa cells and human cervical epithelial cells (HCECs) under different temperature gradients (37–42 ℃ and 60 ℃). Continuous tracking of cell thickness and volume was achieved. The results revealed a unique morphological thermoresponsive process of the cells. The study determined the temperature threshold and exposure duration for high-temperature-induced effects in HeLa cells. The greater temperature sensitivity of HeLa cells compared with HCECs has been verified. This technology is expected to provide an effective means for evaluating morphological changes in cellular thermoresponses, offering novel insights for optimizing personalized cancer treatment regimens.</p>