Hydrological changes significantly affect organic matter export in flood plains, which is a vital aspect of aquatic food webs and biogeochemical cycles. The frequency and duration of floods are projected to increase; thus, the effects of flood duration on labile forms of organic matter that respond quickly to environmental changes need to be investigated. To better understand the effect of flood duration on dissolved organic matter (DOM), we used columns under controlled laboratory conditions to simulate moderate flooding in riparian forest soils. We monitored changes in physicochemical properties and DOM after 1, 3, 8, 16, and 30 days of flooding (FDs) and up to 30 days after flooding (DAF). pH and electrical conductivity (EC) were significantly affected by flood duration; increasing EC and pH were linked to increasing water-extractable organic carbon (WEOC) after 16 FDs and total dissolved nitrogen (TDN) in soil. The variations included decreased protein-like components after 16 and 30 FDs, increased aromaticity, and TDN in the flood water. In post-flooded soil, WEOC varied with flood duration, that is, 16 > 8 > 3 FDs; however, based on the physical, chemical, and DOM attributes, the principal component analysis distinguished the soil into three groups: initial, early days after flooding (approximately 5 DAF), and 30 DAF, with 30 DAF having a higher fluorophore to WEOC ratio. Flood duration and post-flooding period affected DOM directly and indirectly through changes in physical conditions influencing microorganisms while simultaneously fostering exchange between the soil and flood water during periods of inundation.