Two-photon fluorescence microscopy is a widely used noninvasive bioimaging technique because of unique advantages such as a large penetration depth and 3D mapping capability. Ideal two-photon fluorophores require large two-photon absorption cross sections and red emission with high quantum yields. Here we report red-emitting-dye-doped conjugated polymer nanoparticles that display high two-photon excitation brightness. In these nanoparticles, conjugated polymer (PFV) was chosen as a two-photon light-harvesting material, and red-emitting dyes (MgPc and Nile red) were chosen as the energy acceptors and red-emitting materials. Two-photon excitation fluorescence of MgPc and Nile red was enhanced by up to similar to 53 and similar to 240 times, respectively. We have successfully demonstrated the application of these conjugated polymer-based nanoparticles in two-photon excitation cancer cell imaging with an excellent contrast ratio. This concept could become a general approach to the preparation of two-photon excitation red-emitting materials for deep-tissue live-cell imaging with high contrast.