The hump-like anomalous Hall effect (AHE) is an intriguing phenomenon in physics and the material community, with significant potential applications for spintronics. The physical mechanism responsible for this effect is still a topic of debate. Here, the unconventional hump-like AHE is observed in the epitaxial SrRuO 3 (SRO)/Sr 3 Al 2 O 6 (SAO) heterostructure with a relatively thick SRO thickness, accompanying the sign change of anomalous Hall resistance. This hump-like AHE disappears in the freestanding SRO film after the SAO layer was dissolved in water. Additionally, the hump-like feature could be manipulated by the in-plane tensile strain and oxygen vacancies through varying the SAO thickness and in situ oxygen annealing, respectively. A two-domain model, namely, domain I and domain II for negative and positive Hall resistance, is employed to comprehensively understand the hump-like AHE observed in SRO/SAO heterostructures. The positive and negative anomalous Hall resistances are strongly bonded to the intrinsic nontrivial band structure. Microscopically, the formation of the two-domain is closely related to the modulation of Ru−O−Ru bond/length as well as the octahedral rotation. This study provides some comprehensive understanding of the origination of hump-like AHE in SRO-based films, which could facilitate the application of SRO in spintronics.