Microbial fuel cell (MFC) biosensors are self-sustainable device for monitoring of various substrates; however, for heavy metals detection are still scarce. In this study, E. coli BL21 was engineered to express the zntR, ribB, and oprF genes with P promoter, which could sense zinc (Zn) for riboflavin and porin production. The engineered strain produced high levels of riboflavin (2.4-3.6 μM) and improved cell membrane permeability, with a positive correlation of Zn (0-400 μM). The strain was then employed in MFC biosensor under the following operational parameters: external resistance 1000 Ω, pH 9, and temperature 37 °C for Zn sensing. The maximum voltages (160, 183, 260, 292, and 342 mV) of the constructed MFC biosensor have a linear relationship with Zn concentrations (0, 100, 200, 300, and 400 μM, respectively) (R = 0.9777). An Android App was developed for the biosensor system that could sense Zn in real-time and in situ. The biosensor was applied to wastewater with different Zn concentrations and the results showed that the detection range for Zn was 20-100 μM, which covers common Zn safety standards. The results obtained with developed MFC biosensor were comparable to conventional methods such as colorimetric, flame atomic absorption spectroscopy (FAAS), and inductively coupled plasma optical emission spectroscopy (ICP-OES). In summary, MFC biosensor with biosynthetic strain is an efficient and affordable system for real-time monitoring and sensing of heavy metals.