Time-resolved quantitative measurements of formaldehyde (HCHO) in the low-temperature oxidation of iso-octane using a rapid compression machine have been performed with mid-infrared laser absorption spectroscopy. Due to the weak interference of the broadband absorption of iso-octane, a two-color detection scheme was applied to HCHO detection. The cross-sections of HCHO and iso-octane in two colors were measured using the rapid compression machine in the temperature range of 450–737 K and pressure range of 100–700 kPa. The time-resolved quantitative HCHO profiles in the low-temperature oxidation of iso-octane at 0.77 MPa, 645 K, and an equivalence ratio of 1.0 were successfully obtained. The calculated HCHO profiles using the latest chemical kinetic model of iso-octane show the same tendency as the experimental profiles.