Wolframite is often associated with calcium bearing minerals, leading to low grade, while conventional anionic collectors are hard to realize their efficient flotation separation, thus the effective structural modification of cationic collectors provides a new insight into enhancing the comprehensive development of wolframite resources. In this study, a surfactant 3-dodecyloxy propyl amidoxime (DOPA) was designed and first used as the collector in flotation separation of wolframite from fluorite and calcite. Computational calculations basically proved the correctness of predictions about the performances of DOPA. Micro-flotation tests indicated that compared to DDA and conventional anionic collector benzohydroxamic acid (BHA), DOPA possessed superior collecting ability to wolframite and excellent selectivity against fluorite and calcite at low concentration, with no frother or activator employed, which is supposed to be an efficient collector for separating wolframite from calcium bearing minerals. The separation mechanisms of DOPA between wolframite and fluorite/calcite were further probed, revealing that besides the electrostatic attraction between DOPA and wolframite, DOPA could chemisorb onto wolframite surfaces by forming five-membered ring toward the surface cation site Fe or Mn, with no significant interaction observed on fluorite/calcite surfaces, resulting to the excellent separation performance.