Surface water-groundwater (SW-GW) interactions critically regulate the transport and transformation of dissolved organic matter (DOM), yet quantitative linkages remain elusive, especially in semi-arid river systems characterized by frequent hydrological conversion. Combining stable isotope (delta O-18, delta H-2) tracing with DOM fluorescence fingerprinting, we revealed wet-season spatial patterns of SW-GW interactions and their deterministic controls on DOM compositional dynamics in the Ba River. Isotopic mass balance revealed a clear hierarchical pattern in SW-GW interactions along the river continuum: negligible groundwater input (3%) in the upstream, significant groundwater discharge (34%) in the midstream, and a spatially complex pattern of bidirectional water exchange in the downstream. These hydrological transformations drove a systematic evolution in DOM: terrestrial humic-like components prevailed under weak SW-GW connectivity; groundwater discharge introduced abundant microbial protein-like material, raising the biological index (BIX); and bidirectional exchange generated unique hybrid fingerprints. From this coherent response, we identified region-specific fluorescence boundaries (FI = 2.4, BIX = 0.95, HIX = 0.70) that identify SW-GW interactions with accuracy rivaling isotopic tracers (error < 5%). This work establishes DOM fluorescence not merely as a descriptive marker but as a quantitative proxy for hydrological processes, providing a novel framework to mechanistically link water cycling with carbon transformation at aquatic interfaces in semi-arid river systems.