Hypoxia and sediment particle resuspension (SPR) alter the biogeochemical cycle of estuarine and coastal seas, which in turn affects the production and emission of methane (CH4), nitrous oxide (N2O) and carbon dioxide (CO2) greenhouse gases (GHGs) in estuaries. Despite the importance of CH4, N2O and CO2 in estuarine ecosystems, little is known about their magnitude and spatiotemporal variation under the combined influence of hypoxia and SPR. This study utilized continuous mooring observations to investigate the temporal and spatial variations of GHGs before and after hypoxia in the Pearl River Estuary (PRE). The results showed that the concentration of GHGs in the water column increased significantly following hypoxia as compared to its absence. The synergistic effect of SPR and hypoxia significantly enhances GHGs production and accumulation in bottom water. Anaerobic mineralization of organic matter (OM) in an environment with severely low dissolved oxygen (DO) is the primary determinant for increased CH4 concentration, while OM and CH4 oxidation are the main drivers for maintaining high CO2 concentration in subsurface water. Hypoxic development enhanced denitrification N2O production in the water column. The presence of SPR enhanced oxygen-consuming coupled hypoxia significantly stimulated the increase of CH4, N2O and CO2 concentrations in the water column. Hypoxic development results in an increased water–air GHGs flux, but this effect may be masked by runoff plumes with high GHGs concentrations in the regions near the river outlets. This study highlights that hypoxia leads to significant increases in anaerobic GHGs production and subsequent emissions from estuarine water columns.