Photoplethysmography (PPG) signal has been used as an advantageous analytical technique in biological and physiological assessments. Most analyses of PPG signal are based on time and frequency domain characteristics or shape of the signal. In the present study, time difference variations between two separate simultaneous PPG signals were extracted and proposed as a new strategy for the analysis of two-wavelength PPG. A dual-channel PPG biosensor was applied to obtain simultaneous long-term infrared and red transmission mode PPG signals. Then, time differences between the peak points of the obtained PPG pulses were extracted in each beat of the heart for various physiological conditions monitoring. The proposed method was verified in three different physiological changing situations (wakefulness-sleep, rest-run, nonsmoking-smoking) with the participation of ten healthy individuals in two iterations. Results showed that different PPG signals had significant time difference variations (time shifts relative to each other) with changes in a psychophysical state of investigated subjects. This earlier unreported phenomenon would have been a novel and interesting finding.