Background: In exercise testing, the ventilatory threshold 1 (VT1) and ventilatory threshold 2 (VT2) are used in lifestyle-related diseases, cardiac rehabilitation, and athletic training. We investigated a VT2 measuring method using a pulse oximeter. Methods: Thirty-four adults (men: 15; women: 19) performed a bicycle ergometer Ramp Test. VT1 values were determined using expiratory gas data. The bifurcation of the curve obtained by designating the pulse rate (PR) as an independent variable and SpO₂/PR as a dependent variable was calculated using the residual sum of squares and defined as the SpO₂ threshold (ST) (SpO₂-Slope method). A second bifurcation with ST as the origin was further defined (ST2). ST2 validity was assessed by comparing and analyzing the differences and correlations with each VT2 obtained by expiratory gas analysis. Results: The correlation between ST2 determined by the SpO₂-Slope method using PR as an index and VT2 obtained from respiratory gas analysis was significant, showing a positive correlation (r = 0.74~0.92; p < 0.01), with most data points falling within the 1.96 ± SD in the Bland–Altman analysis. Conclusions: ST2 values derived from SpO₂ and pulse rate measurements by pulse oximeter may be a valuable VT2 measuring method.