The design of the light environment plays a critical role in the interaction between people and visual objects in space. Adjusting the space’s light environment to regulate emotional experience is more practical for the observers under lighting conditions. Although lighting plays a vital role in spatial design, the effects of colored lights on individuals’ emotional experiences are still unclear. This study combined physiological signal (galvanic skin response (GSR) and electrocardiography (ECG)) measurements and subjective assessments to detect the changes in the mood states of observers under four sets of lighting conditions (green, blue, red, and yellow). At the same time, two sets of abstract and realistic images were designed to discuss the relationship between light and visual objects and their influence on individuals’ impressions. The results showed that different light colors significantly affected mood, with red light having the most substantial emotional arousal, then blue and green. In addition, GSR and ECG measurements were significantly correlated with impressions evaluation results of interest, comprehension, imagination, and feelings in subjective evaluation. Therefore, this study explores the feasibility of combining the measurement of GSR and ECG signals with subjective evaluations as an experimental method of light, mood, and impressions, which provided empirical evidence for regulating individuals’ emotional experiences.