Phytoplankton contribute almost half of the world's total primary production. The exudates and viral lysates of phytoplankton are two important forms of dissolved organic matter (DOM) in aquatic environments and fuel heterotrophic prokaryotic metabolism. However, the effect of viral infection on the composition and biological availability of phytoplankton-released DOM is poorly understood. Here, we investigated the optical characteristics and microbial utilization of the exudates and viral lysates of the ecologically important unicellular picophytoplankton Prochlorococcus Our results showed that Prochlorococcus DOM produced by viral lysis (Pro-vDOM) with phages of three different morphotypes (myovirus P-HM2, siphovirus P-HS2 and podovirus P-SSP7) had higher humic-like fluorescence intensities, lower absorption coefficients and higher spectral slopes compared to DOM exuded by Prochlorococcus (Pro-exudate). The results indicate that viral infection altered the composition of Prochlorococcus-derived DOM and might contribute to the pool of oceanic humic-like DOM. Incubation with Pro-vDOM resulted in a greater dissolved organic carbon (DOC) degradation rate and decreases in the absorption spectral slope and heterotrophic bacterial growth rate compared to incubation with Pro-exudate, suggesting that Pro-vDOM was more bioavailable compared to Pro-exudate. In addition, the stimulated microbial community succession trajectories were significantly different between the Pro-exudate and Pro-vDOM treatments, indicating that viral lysates play an important role in shaping the heterotrophic bacterial community. Our study demonstrated that viral lysis altered the chemical composition and biological availability of DOM derived from Prochlorococcus, which is the numerically dominant phytoplankton in the oligotrophic ocean.Importance The unicellular picocyanobacterium Prochlorococcus is the numerically dominate phytoplankton in the oligotrophic ocean, contributing to the vast majority of marine primary production. Prochlorococcus releases a significant fraction of fixed organic matter into surrounding environment and supports a vital portion of heterotrophic bacterial activity. Viral lysis is an important biomass loss process of Prochlorococcus Yet little is known about whether and how viral lysis affects Prochlorococcus-released dissolved organic matter (DOM). Our paper shows that viral infection alters the optical properties (such as the absorption coefficients, spectral slopes and fluorescence intensities) of released DOM and might contribute to a humic-like DOM pool and carbon sequestration in the ocean. Meanwhile, viral lysis also releases various intracellular labile DOM including amino acids, protein-like DOM and lower-molecular weight DOM, increases the bioavailability of DOM and shapes the successive trajectory of the heterotrophic bacterial community. Our study highlights the importance of viruses in impacting the DOM quality in the ocean.