Mesoscale eddies have great influence on heat and material transport in the ocean and thus play an important role in modulating the global climate variability. However, our understanding of their fine three-dimensional (3D) thermohaline and biogeochemical structure remains incomplete because of the scarcity of high-resolution measurements. This research aims to construct the fine 3D structure of an anticyclonic eddy in the northern South China Sea (NSCS) to validate the effectiveness of a glider network for observing mesoscale eddies. Twelve Petrel gliders were deployed in NSCS during August 2017 to gather fine information of an anticyclonic eddy. By combining the high-resolution in situ glider data and the sea level anomaly data, we have constructed a detailed 3D structure of the eddy. The analysis results of the absolute dynamic topography map and the water mass comparison imply that the anticyclonic eddy may generate from eddy shedding of the Kuroshio loop current. The maximum potential temperature anomaly (over 3°C) appears at the thermocline, and the maximum salinity anomaly (~0.8 psu) exists at ~50 m. The maximum value of the dissolved oxygen concentration (~7.5 mg L−1) appears at 50–80 m. The maximum chlorophyll concentration (~1.2 μg L−1) lies at 80–120 m, just below that of the dissolved oxygen concentration. The colored dissolved organic matter concentration increases with depth, with the mean value being less than 1.23 ppb above 50 m and beyond 2.2 ppb at 500–800 m. The results verify the capability of the glider network to observe a fine-scaled 3D structure of mesoscale eddies and will provide a useful guide for future eddy observations.