Johor Strait is a narrow strait located between Malaysia and Singapore; and serves as the main marine aquaculture area for the two countries. The Strait was subjected to anthropogenic activities that led to eutrophication and an increase in harmful algal bloom (HAB) with damages to the aquaculture industry of both Malaysia and Singapore.  In this study, the occurrence of high biomass algal blooms and hypoxia condition along the inner part of Johor Strait was investigated as an effort to continuously monitor the water quality and occurrence of harmful algal blooms. Monthly vertical profiles of physical water parameters (temperature, salinity, and dissolved oxygen) were measured in situ at different depths (subsurface, 1 m, 5 m, and 8 m) depending on the depth of the water column at the sampling stations between May 2018 and April 2019. The spatial-temporal variability of macronutrients and chlorophyll-a level was analysed. Our results showed that high chlorophyll-a concentration (up to 48.8 μg/L) and high biomass blooms of Skeletonema, Chaetoceros, Rhizosolenia, and Thalassiosira were observed seasonally at the inner part of the strait. A hypoxic to anoxic dead zone, with dissolved oxygen levels ranging from 0.19 to 1.7 mg/L, was identified in the inner Johor Strait, covering an estimated area of 10.3 km². Prolonged high biomass of diatom blooms triggered by eutrophication were the factors that induced the formation of hypoxic-anoxic zones in the inner part of Johor Strait.