The nanoscale organization and corresponding electronic properties of a photoactive donor-acceptor blend layers based on regioregular poly (3-hexylthiophene) (RR-P3HT) donor with fullerene and non-fullerene acceptors on Au(111) substrate has been studied using scanning tunneling microscopy and spectroscopy (STM/STS). Subsequent to annealing treatment, STM topography and dI⁄dV images are observed as a combination of phase-separated donor-rich, acceptor-rich, and mixed donor-acceptor domains. This technique permits to explore simultaneously the quantitative linkage between the nanoscale morphologies and corresponding local electronic properties. We determine the HOMO and LUMO-edges at the individual domains and interfacial band alignments of the donor-acceptor interface. We have observed a noteworthy deeper HOMO energy of RR-P3HT in mixed-region associated primarily with the degree of disorder-induced band gap widening of the polymer and donor:acceptor intermolecular interactions. Similarly, LUMO of the acceptor in the mixed region is also raised due to intermolecular interaction. These energetic difference in the mixed phase is likely to be responsible for the reduced recombination in bulk heterojunction (BHJ). Hence, this characterization provides nanoscale insight to the annealing-induced morphological organization and corresponding local electronic properties account for an impressive increase of the charge generation, transport and corresponding device performance of the BHJ solar cells.