How to on-line analyze the morphology of composite polymer particles is a great challenge to develop the high-performed polymer nanoparticle materials, not only academia aspect but also industrial applications. We aim to on-line detect the weight fraction of each phase in composite polymer particles with a multilayered structure during emulsion polymerization. This work is also hoped to contribute to the development of on-line analysis on the morphology of composite polymer particles in the future. The weight fractions of core phase, interfacial layer, and shell layer (Wc, Wi, and Ws, respectively) in the multilayered composite polymer particles prepared by semi-continuous seeded emulsion terpolymerization of styrene (St), butyl acrylate (BA), and methyl methacrylate (MMA) with the existence of St-BA copolymer seed particles were successfully analyzed by using on-line near-infrared (NIR) spectroscopy for the first time on the basis of the results obtained by multi-frequency temperature-modulated differential scanning calorimetry (TOPEM-DSC), where NIR calibration models on Wc, Wi, and Ws were successfully established. By using NIR spectroscopy and the established models, the values of Wc, Wi, and Ws could be accurately on-line detected. It is suggested that there is a possibility to estimate the formation of unique morphology in composite polymer particles by on-line NIR measurement.