Oceanic dissolved organic matter (DOM) comprises a complex molecular mixture which is typically refractory and homogenous in the deep layers of the ocean. Though the refractory nature of deep-sea DOM is increasingly attributed to microbial metabolism, it remains unexplored whether ubiquitous microbial metabolism of distinct carbon substrates could lead to similar molecular composition of refractory DOM. Here, we conducted microbial incubation experiments using four typically bioavailable substrates (L-alanine, trehalose, sediment DOM extract, and diatom lysate) to investigate how exogenous substrates are transformed by a natural microbial assemblage. The results showed that although each-substrate-amendment induced different changes in the initial microbial assemblage and the amended substrates were almost depleted after 90 days of dark incubation, the bacterial community compositions became similar in all incubations on day 90. Correspondingly, revealed by ultra-high resolution mass spectrometry, molecular composition of DOM in all incubations became compositionally consistent with recalcitrant DOM and similar toward that of DOM from the deep-sea. These results indicate that while the composition of natural microbial communities can shift with substrate exposures, long-term microbial transformation of distinct substrates can ultimately lead to a similar refractory DOM composition. These findings provide an explanation for the homogeneous and refractory features of deep-sea DOM.