Japanese tomato cultivar ‘Rinka 409’ is suitable for harvesting at a fully ripe stage since it remains firm at the stage, resulting in longer shelf life. However, it is difficult to estimate the quality change because its color does not change after the fully ripe stage. For ‘Momotaro,’ we previously proposed a fluorescence-based indicator of overripening; however, its suitability for another cultivar, ‘Rinka 409’, and its relationship with temperature and quality were unclear. Therefore, in this study, we investigated the appropriateness of this fluorescence-based index for the above purposes. In 2021, 45 tomato seedlings planted in 2020 were harvested at the fully ripe stage and used for experiments. Weight loss and firmness were evaluated to determine the quality of the tomatoes. The post-harvest temperatures were set as 10, 17, and 25 °C. The results showed that the rate of weight loss increased linearly, whereas the firmness decreased exponentially. For non-destructive estimation of these properties, the excitation-emission matrix (EEM) was measured in the range of 250–460 nm and 280–750 nm for the excitation and emission, respectively. ‘Rinka 409’ showed a strong fluorescence emission at 400 nm/620 nm of the excitation/emission, respectively, regardless of days and temperature. The fluorescence intensity, as well as the firmness, decreased exponentially. Thus, a linear relationship was observed between them. The firmness of ‘Rinka 409’ can be estimated using a simpler linear regression model using this method than previous researches.