We experimentally realized one-scan fluorescence emission difference nanoscopy (FED) by simultaneously imaging two different color emissions of NaYF4:Er3+@NaYF4@NaYF4:Yb3+/Tm3+ upconversion nanoparticles. Under the irradiation of two synchronized laser beams, a solid 940 nm beam and a hollow 808 nm beam, green emission of Er3+ and blue emission of Tm3+ can be orthogonally generated and collected. After simple subtraction, a resulting super-resolution image featuring 54 nm resolution was obtained. This strategy of excitation orthogonality would greatly improve the imaging speed and the applicability of FED nanoscopy.