Abstract: As an important sink for silver nanoparticles (AgNPs), sediments are also the source of contaminated AgNPs in the aquatic environment, however, there is a serious lack of understanding about the release of AgNPs in sediments and their environmental risks. Natural organic matter (NOM), as an important component of sediments, how to affect the release of AgNPs in sediments is still unclear. This study used humic acid (HA) as NOM to investigate the effect of different HA contents (0, 1%, 5% and 10%) on the release of AgNPs from sediments (AgNPs concentration is 100 μg/g Ag dw) and the toxic effects of released Ag on zebrafish. The results showed that the concentration of total Ag in the water column was (0.01±0.01)—(2.53±0.52) μg/L after 15 and 30 days of exposure, and the released Ag concentration from high HA content group (5% and 10%) was higher than that of low HA content group (0 and 1%), indicating that HA promoted AgNPs release from the sediment. The released AgNPs can accumulate in zebrafish tissues (mainly in the liver and intestine) to cause tissue oxidative stress. Ag also impact the microbial community structure in the zebrafish intestine based on 16S rRNA high-throughput sequencing results. The study proved the release rule of AgNPs in sediments and its environmental risk, which can provide an important basis for comprehensive evaluation of environmental behavior and ecological safety of AgNPs.