Fluorite and scheelite, which are strategic calcium-bearing minerals, have similar active sites (Ca2+); as a result, the efficient separation of the two minerals is still one of the world’s most difficult problems in the field of flotation. In this work, N-decanoylsarcosine sodium (SDAA), a non-toxic and low-cost amino acid surfactant, was applied in the flotation separation of fluorite from scheelite for the first time. In the test, single mineral, binary mixed minerals, and actual ore experiments showed that the pre-removal of fluorite from scheelite by reverse flotation can be achieved. The results of adsorption capacity detections, zeta potential tests, and FTIR analysis showed that the negatively charged SDAA prefers to adsorb onto the positively charged fluorite surface due to the electrostatic interaction. The results of crystal chemistry and DFT calculations showed that SDAA has a stronger chemical interaction and more electron transfer numbers to the Ca atom on the fluorite surface and forms a Ca-SDAA complex. Therefore, the significant difference in the adsorption behavior of SDAA on the surfaces of two minerals provided a new insight into the separation efficiency of amino acids and possesses a great potential for industrial application in scheelite flotation.