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Mesoscale eddies play an important role in modulating the ocean circulation. Many previous studies on the three-dimensional structure of mesoscale eddies were mainly based on composite analysis, and there are few targeted observations for individual eddies. A cyclonic eddy surveyed during an oceanographic cruise in the Northwest Pacific Ocean is investigated in this study. The three-dimensional structure of this cyclonic eddy is revealed by observations and simulated by the four-dimensional variational data assimilation (4DVAR) system combined with the Regional Ocean Modeling System. The observation and assimilation results together present the characteristics of the cyclonic eddy. The cold eddy has an obvious dual-core structure of temperature anomaly. One core is at 50–150 m and another is at 300–550 m, which both have the average temperature anomaly of approximately −3.5°C. The salinity anomaly core is between 250 m and 500 m, which is approximately −0.3. The horizontal velocity structure is axis-asymmetric and it is enhanced on the eastern side of the cold eddy. In the assimilation experiment, sea level anomaly, sea surface temperature, and in situ measurements are assimilated into the system, and the results of assimilation are close to the observations. Based on the high-resolution assimilation output results, the study also diagnoses the vertical velocity in the mesoscale eddy, which reaches the maximum of approximately 10 m/d. The larger vertical velocity is found to be distributed in the range of 0.5 to 1 time of the normalized radius of the eddy. The validation of the simulation result shows that the 4DVAR method is effective to reconstruct the three-dimensional structure of mesoscale eddy and the research is an application to study the mesoscale eddy in the Northwest Pacific by combining observation and assimilation methods. [ABSTRACT FROM AUTHOR]