Sports injuries often arise from improper scheduling of exercise loads, and timely assessment of these loads is essential for minimizing injury risk. This article investigates the validity of using changes in pupillary light reflex (PLR) during dynamic aerobic training as a novel approach to evaluating exercise load. Dynamic aerobic training was conducted on a power bicycle for 15 min. With a 3-minute interval as the demarcation line, PLR measurement was performed before training and heart rate was recorded throughout the process. The Rating of Perceived Exertion (RPE) scale invented by Borg was used for scoring before training and after training. The normal distribution of data was confirmed through the Shapiro-Wilk test. Pearson correlation analysis was used to quantify the correlation between variables. The Receiver Operating Characteristic (ROC) curve and the area under the curve (AUC) analysis were used to determine the indicators of exercise load. The optimal threshold of the indicators was calculated through the Youden index to evaluate sensitivity and specificity. Thirty male second-tier athletes with a mean age of 23.66 ± 2.21 years, a mean height of 175.3 ± 6.5 cm, and a mean weight of 68.99 ± 10.35 kg participated in this study. Based on the RPE scale results, it was confirmed that the 15-minute dynamic aerobic exercise successfully elicited varying levels of perceived exertion among the athletes. The findings of this study indicate significant changes in PLR and heart rate (HR) with increasing exercise duration and external load. There were strong correlations between RPE and maximum constriction velocity (MCV) (|r| = 0.8309, p < 0.001, negative correlation), maximum diameter (INIT) (r = 0.7641, p < 0.001, positive correlation), time to reach 75% recovery (T75) (|r| = 0.7289, p < 0.001, negative correlation), and HR (r = 0.8170, p < 0.001, positive correlation). Additionally, the results suggest that MCV is the most significant potential indicator for detecting internal load, exhibiting high specificity and sensitivity (AUC = 0.8509, p < 0.001). Further analysis using the Youden index identified 5.07 mm/s as the optimal cutoff value for MCV, indicating that when MCV ≤ 5.07 mm/s, the athletes’ internal load has reached an “Intense” state. PLR may be a potential indicator for assessing internal load Further investigation could involve developing a non-invasive exercise load detection system based on pupillary variable indicators, providing a valuable new approach for accurately measuring exercise load.