A new biological assessment technique was presented by novel use of photoplethysmography (PPG) signals. Most previous PPG signal analysis techniques were based on morphological and frequency characteristics of PPG. In the present study, time differences (TDs) between separate pairs of PPG signal (with different wavelengths) were used as the novel aspect of PPG technology. A computer connectable multi-channel PPG device and a MATLAB ® based program were used to record transmission mode PPG signals with wavelengths of 940 nm (IR), 660 nm (red), 520 nm (green) and 450 nm (blue). The TDs between three different pairs of the signals were obtained during a breath holding experiment (as biology challenge test) to investigate and compare their Time Difference (TD) variations within the tests. The experiments were performed with the participation of 25 healthy subjects. Results showed that TDs of PPG signal pairs had significant variations (p<0.05) in reply to biological challenges. It was concluded that TD variations can be indicative of very important biological information. A good attribute of PPG TD method was its independence from signal amplitude and avoiding from corresponding disadvantage.