While most of the studies focused on microplastic (MP) pollution in both marine and continental environments, estuaries are poorly documented and their role as an important pathway and a physical filter are still unclear. In this study, the fate of MP was investigated along the salinity gradient of the Seine well-mixed estuary and over two tidal cycles. Four sampling surveys were conducted at three stations during the same day (La Bouille-upper estuary, Vieux-Port-middle estuary and La Roque-lower estuary) along the salinity gradient. For each survey, surface water (first 15 cm including the sea surface microlayer - SML) and sub-surface water (first 50 cm excluding SML) were collected using a 300-µm mesh size manta net during the ebb tide. Fragments and microbeads were considered while microfiber pollution was excluded. Concentrations in surface waters were 5 to 6 times higher than those in subsurface waters, due to the significant accumulation of MP in SML. Differences were observed along the salinity gradient with the highest concentrations measured at Vieux-Port station located upstream the maximum turbidity front (MTF) and lower concentrations at La Bouille and La Roque stations. This could be explained by i) the salt intrusion and ii) the accumulation of macrolitters in the vicinity of Vieux-Port. Two tidal cycles were also investigated in La Roque, which is characterized by the presence of the MTF. For both cycles, the vertical distribution was examined by sampling sub-surface water, middle-water column and bottom layer during flood and ebb tides. Results clearly demonstrated a vertical gradient between water collected at the bottom (6 and 10 m) exhibiting the highest concentrations while lowest values were observed in surface water. This gradient can be explained by both the resuspension of bottom sediments, rich in MP, during the flood tide and sedimentation during the long water high period and the ebb tide.