Abstract: During focused ultrasound hyperthermia, the precise measurement and assessment of the internal focal temperature within tissues is of significant importance for treatment. To realize non-contact measurement of the internal focal temperature during ultrasound application, a scheme based on inverting the post-focal transmitted acoustic pressure along the acoustic axis was proposed. Combined with the simulation of acoustic field and thermal field coupling theory, an experimental protocol for non-invasive assessment of the internal focal temperature in tissues during ultrasound hyperthermia was formulated. Experiments were carried out using gel phantoms, fresh fat, pork tenderloin, and other tissues. The results indicated that, compared with the simulation-calculated results, the average errors of the method by detecting the post-focal transmitted acoustic pressure were 6.25%, 1.53%, and 8.13%, respectively; compared with the results from thermocouple verification experiments, the average errors were 8.30%, 7.56%, and 9.15%, respectively. The results were well-matched, and non-contact measurement of the internal focal temperature could be achieved without protein denaturation in tissues. This provides a basis for planning the dosage administration of ultrasound therapy and holds clinical application prospects.