Photochromism is a light-induced reversible or irreversible transformation in chemical species between two forms having different absorption spectra. Photochromic materials have been the focus of intensive investigations for several decades because they have high potential for applications to optically rewritable data storage, optical switching, and chemical sensing. Like other stimuli-responsive materials such as heat and pH-sensitive materials, photochromic devices require that either the nanostructure or the molecular structure be responsive to an external stimulus—in this case, light. We reported that polymer nanoparticles formed by emulsion polymerization have photochromic effects; in particular, the fluorescence of these photochromic nanoparticles can be optically switched “on” and “off” with specific wavelengths of UV or visible light. However, in biological applications, fluorescence switching should be not only reversible and high contrast so that it can be used to selectively highlight cells, organelles, or proteins, but also UV irradiation should be avoided in utilization. Therefore, two-photon excitation is prefered because it could decrease cellular and tissue damage. We propose that spiropyran-containing nanoparticles (SP), which are switched using light with the wavelength lower than 420 nm, can also be activated using near-IR lower than 800nm through two-photon absorption and excitation process. That means, the photoisomerization induced by two-photon absorption (TPA) of 780 nm wavelength of femtosecond pulses causes not only the photochromism but also the photoswitchable fluorescence of nanoparticles. Here, we use two-photon near-IR laser source to induce the photoisomerization of spiropyrans in the nanoparticles and successive two-photon excitation fluorescence imaging. It is demonstrated that the cellular fluorescence imaging using the SP nanoparticle-anti-Her 2 monocloned antibody conjugates to target the Her2 in the surface of human breast cancer cell line SK-BR-3 is optically switchable.