Phlegmacins are homodimeric dihydroanthracenone natural products featuring two torosachrysone monomers unsymmetrically conjugated by 7,10'-coupling. Herein, we report the identification and characterization of the biosynthetic gene cluster of phlegmacins in ascomycete Talaromyces sp. F08Z-0631. On the basis of the heterologous reconstitution of the phlegmacin pathway in Aspergillus oryzae, we demonstrated an unprecedented laccase-involved unsymmetrically regioselective oxidative coupling reaction. The association of laccase PhlC and the fasciclin partner protein PhlB was verified to be indispensable for the coupling activity. Intriguingly, both proteins can be transferred and located independently at the mitochondrial membrane. Notably, only their subcellular colocalization led to the occurrence of oxidative dimerization. These observations add new insights into the poorly understood catalytic mechanisms of various laccases involved in the biosynthesis of secondary metabolites, particularly those functioning with variable partners.