It is challenging to achieve deep microscopic imaging for the strong scattering in biotissue. An efficient three-photon luminescence can effectively increase the penetration depth. Here we report that β-NaYF4: Er3+/NaYF4 UCNPs were excited by a 1480-nm CW-laser and emitted 543/653-nm light through a three-photon process. With the merit of the hexagonal crystal phase, sub-milliwatt laser power was utilized to excite the UCNP-probed cells to minimize the heating effect. The polymer-coated UCNPs were shown to be harmless to cells. The deep, high contrast in vitro microscopic imaging was implemented through an artificial phantom. Imaging depth of 800 μm was achieved using only 1.5 mW excitation and a 0.7 NA objective. The green/red emission intensities ratio after penetrating the phantom was studied, indicating that longer emission wavelength is preferred for deep multiphoton microscopy. The proposed and demonstrated β-UCNPs would have great potential in three-photon microscopy.