Flotation reagents can change the surface properties of minerals, leading to differences in the interaction between mineral particles and affecting the mutual aggregation or dispersion of particles. In this work, we studied the role of activator copper sulfate, collector butyl xanthate and frother terpineol in adjusting the potential energy of pyrite particles from the perspective of the interfacial interaction. We evaluated the surface characteristics using contact angle analysis and zeta potential measurements under different reagents. A microscope was used to observe aggregation state of particles. The hydrophobic agglomeration kinetics of pyrite was studied through the turbidity meter measurement, and the interaction energy between pyrite particles was calculated using the extended-Derjaguin-Landau-Verwey-Overbeek (extended-DLVO) theory. The results showed that the repulsive potential energy is dominant among pyrite particles in aqueous suspensions and that the particles are easy to disperse. Flotation reagents can effectively reduce the repulsive energy between pyrite particles and increase the attraction energy between particles, which is conducive to the hydrophobic agglomeration of fine pyrite. Reagent molecules can greatly reduce the electrostatic repulsion potential energy of the pyrite particles’ interface, increase the hydrophobic attraction potential energy between the particle interfaces, and its size is 2 orders of magnitude larger than the van der Waals attraction potential energy, which is the main reason for induced the agglomeration of fine pyrite and is conducive to the flotation recovery of fine pyrite. Generally, the order in which the reduction of pyrite agglomeration was affected by the additions of flotation reagents was butyl xanthate > terpineol > copper sulfate.