Herein, 2D/2D porous Co3O4 nanosheets/rGO composites modified GCE (Co3O4/rGO/GCE) were proposed for electrochemical detection of tryptophan (Trp). The surface morphology and structure of Co3O4/rGO nanosheets were carried out using SEM, XRD, and Raman spectra. Electrochemical performance of Trp on as-synthesized modified electrodes were discussed by CV and EIS techniques. Moreover, the best electrochemical property was also investigated by varying test condition detailedly. Consequently, oxidation peak intensity and current density were 130.3 μA and 336.7 μA·cm−2 on Co3O4/rGO/GCE at working potential of 0.78 V (vs. SCE electrode), respectively. They were more than 23-times greater than these on bare GCE in electrochemical detection of Trp. It was attributed to the synergy effect of 2D porous Co3O4 nanosheets (high catalytic activity) and 2D rGO nanosheets (large electrochemical active area). After the optimization, wide linear determination ranges (from 1.0 to 800 μM) and low LOD of 0.26 μM (S/N = 3) were acquired. In addition, these electrodes were further successfully applied to the determination of Trp in commercial amino acid injection samples. This 2D porous Co3O4/rGO modified GCE sensor could provide a slight view on the design of 2D/2D composite materials with high electrochemical properties.