For the first time, this study explored spatio-temporal variation in water quality and phytoplankton community structure in Changwang, Meishe, and Wuyuan Rivers in tropical Hainan Island, China. Phytoplankton samples and water were collected between March and December 2019 and analyzed using standard methods. Two-way ANOVA revealed significant spatial and seasonal variation in physico-chemical parameters (p < 0.05). Wuyuan had high TP (0.06 ± 0.04 mg L⁻¹), TN (1.14 ± 0.71 mg L⁻¹), NH4⁺-N (0.07 ± 0.09 mg L⁻¹), Secchi depth (2.28 ± 3.79 m), salinity (3.60±5.50 ppt), and EC (332.50 ± 219.10 µS cm⁻¹). At the same time, Meishe had high TP (0.07 ± 0.03 mg L⁻¹), TN (1.04 ± 0.74 mg L⁻¹), NH4⁺-N (0.07 ± 0.10 mg L⁻¹), EC (327.61 ± 63.22 µS cm⁻¹), and turbidity (40.25 ± 21.16 NTU). In terms of seasons, spring recorded high average TP, TN, NH4⁺-N, COD, and DO, while summer had a high temperature, Chl-a, salinity, and EC. Generally, the physico-chemical parameters met the China water quality standard limits (GB 3838–2002). Overall, 197 phytoplankton species belonging to Cyanophyta, Chlorophyta, Cryptophyta, Bacillariophyta, Pyrrophyta, Euglenophyta, Xanthophyta, and Chrysophyta were identified, with Cyanophyta being dominant. Phytoplankton density showed spatial changes varying from 18 × 10⁶ cell L⁻¹ to 84 × 10⁶ cell L⁻¹. The phytoplankton diversity ranged from 1.86 to 2.41, indicating a mesotrophic state. One-way ANOSIM showed no significant spatial dissimilarity in phytoplankton composition (R = 0.042, p = 0.771) but indicated a significant seasonal difference (R = 0.265, p = 0.001). Therefore, SIMPER analysis revealed that Lyngbya attenuata, Merismopedia tenuissima, Cyclotella sp., Merismopedia glauca, Merismopedia elegans, and Phormidium tenue contributed to the seasonal differences. Furthermore, CCA demonstrated that TP, TN, NH4⁺-N, COD, Chl-a, and Secchi depth greatly influenced the phytoplankton community. This study shows the spatio-temporal variation in water quality and phytoplankton communities, useful for managing riverine quality.