As time plays a fundamental role in our social activities, scholars have studied temporal perception since the earliest days of experimental psychology. Since the 1960s, the ubiquity of color has been driving research on the potential effects of the colors red and blue on temporal perception and on its underlying mechanism. However, the results have been inconsistent, which could be attributed to the difficulty of controlling physical properties such as hue and luminance within and between studies. Therefore, we conducted a two-interval duration-discrimination task to evaluate the perceived duration of color stimuli under different equiluminant conditions: subjective or pupillary light reflex (PLR)-based equiluminance. The results, based on psychometric functional analyses and simultaneous pupillary recordings, showed that the perceived duration of red was overestimated compared with blue even when the intensity of the stimulus was controlled based on subjective equiluminance (Experiment 1). However, since blue is known to induce a larger PLR than red despite equiluminance, we conducted a controlled study to distinguish the indirect effect of pupillary response to temporal perception. Interestingly, the effect observed in Experiment 1 faded when the luminance levels of the two stimuli were matched based on PLR response (Experiment 2). These results indicate that duration judgement can be affected not only by the hue but also by different equiluminance methods. Furthermore, this causality between the equiluminance method and temporal perception can be explained by the fluctuations in incident light entering the pupil.