The separation and enrichment of scheelite from calcite are hindered by the similar active Ca2+ sites of scheelite and the calcite with calciferous gangue. Herein, a novel trisiloxane surfactant, N-(2-aminoethyl)-3-aminopropyltrisiloxane (AATS), was first explored and synthesized and recommended as the collector for the flotation separation of scheelite from calcite. The micro-flotation and mixed binary mineral flotation tests showed that AATS had excellent collection performance for scheelite and high selectivity for calcite within a wide pH range. At the same time, contact angle and zeta-potential measurements, Fourier transform infrared (FTIR) analysis, and density functional theory (DFT) calculations revealed the relevant adsorption mechanism. The contact angle measurement showed that AATS can increase the contact angle of the scheelite surface from 41.7 to 95.8°, greatly enhancing the hydrophobicity of the mineral surface. The results of FTIR analysis and zeta-potential measurement explained that AATS was electrostatically adsorbed on the mineral surface, and DFT calculation further verified that the -N+H3-positive group in AATS was adsorbed on the negatively charged scheelite surface. Therefore, AATS can realize the expectation of high efficiency and selectivity of minerals and enhance the adhesion between the surface of scheelite minerals and bubbles, providing a fresh approach to industrial production.