In this study, a composite film was developed for electrochemical sensing of tryptophan (Trp). Porous reduced graphene oxide (PrGO) was utilized as the electron transfer layer, and a C-undecylcalix[4] resorcinarene Langmuir-Blodgett (CUCR-LB) film served as the molecular recognition layer. Atomic force microscopy (AFM), transmission electron microscopy (TEM), Raman spectroscopy, scanning electron microscopy (SEM), and electrochemical experiments were employed to analyze the characteristics of the CUCR-LB/PrGO composite film. The electrochemical behavior of Trp on the CUCR-LB/PrGO composite film was investigated, revealing a linear response range of Trp was 1.0×10-7 to 3.0×10-5 mol L-1 and a detection limit of 3.0×10-8 mol L-1. Furthermore, the developed electroanalytical method successfully determined Trp content in an amino acid injection samples. This study not only introduces a rapid and reliable electrochemical method for determination of Trp but also presents a new strategy for constructing high-performance electrochemical sensing platforms.