The rapid increase in the usage of plastics globally cause a significant amount of nonrecycled, landfilled and reused plastics released into the environment and accumulated. Identification of microplastics in complex environmental matrices remains a challenge. In this study, we engineered a whole-cell biosensor based on the quorum sensing regulatory circuit LasI-LasR in P. aeruginosa with a reporting module that expressed a LacZ gene. The detection and quantification of blue β-galactosidase activity after incubation in an 5-bromo-4-chloro-3-indolyl-b-galactoside (X-gal) solution act as a visible readout for the amount of quorum sensing signalling molecules, AHLs which ultimately can be used for rapid, sensitive and quantitative detection of microplastics. The results demonstrated that our engineered biosensor pSB1C3-LasR-pLasR3-LacZ was highly sensitive to the AHLs with EC50 values as low as 1.0 X 10-8 M with N-3-oxo-decanoyl L-homoserine lactone (C10-HSL) and 3.2 X 10-8 M with N-3-oxo-dodecanoyl L-homoserine lactone (3OC12-HSL) upon 2 h incubation. Both EC50 values lie within the average range of AHL concentrations in water environments indicating the feasibility of the engineered biosensor in detecting the AHLs. Besides, the engineered biosensor is found to be functional in the real tap water environment and seawater without significant matrix effect which affects the real sample analysis. In addition, we engineered negative control biosensors by removing the sensing module and they were proved to effectively reduce background expression of β-galactosidase activity. Moreover, we developed a standardized protocol of quantitative X-gal assay which can be used as a testing kit in the lab to ensure data consistency for global comparison about microplastic pollution in marine waters. In the protocol, a visible blue colour will be developed in 1 h and the total experiment time is less than 6 h after preparation of overnight culture of biosensor cells. Therefore, our engineered biosensor has potentials to allow rapid, sensitive and high throughput microplastic detection in water samples.