The world's longest trans-basin water diversion project, the Middle-Route (MR) of the South-to-North Water Diversion Project of China (SNWDPC), has officially been in operation for over 5 years since December 2014. Its water quality status has always attracted special attention because it is related to the health and safety of more than 58 million people and the integrity of an ecosystem covering more than 155,000 km². This study presented and analysed the spatio-temporal variations and trends of 16 water quality parameters, including pH, water temperature (WT), dissolved oxygen (DO), permanganate index (PI), five-day biochemical oxygen demand (BOD5), fecal coliform (F. coli), total phosphorus (TP), total nitrogen (TN), ammonia nitrogen (NH3⁻N), sulphate (SO4²⁻), fluoride (F⁻), mercury (Hg), arsenic (As), selenium (Se), copper (Cu), and zinc (Zn), which were determined monthly from samples collected at 27 water quality monitoring stations in the MR of the SNWDPC from March 2016 to February 2019. The water quality index (WQI) was used to evaluate the seasonal and spatial water quality changes during the monitoring period, and a new WQImin model consisting of five crucial parameters, i.e., TP, F. coli, Hg, WT, and DO, was built by using stepwise multiple linear regression analysis. The results demonstrated that the water quality status of the MR of the SNWDPC has been steadily maintained at an “excellent” level during the monitoring period, with an overall average WQI value of 90.39 and twelve seasonal mean WQI values ranging from 87.67 to 91.82. The proposed WQImin model that uses the selected five key parameters and the weights of those parameters has exhibited excellent performance in the water quality assessment of the project, with the coefficient of determination (R²), Root Mean Square Error (RMSE), and Percentage Error (PE) values of 0.901, 2.21, 1.93%, respectively, showing that the proposed WQImin model is a useful and efficient tool to evaluate and manage the water quality. For the management department, the risk sources near certain stations with abnormally high values should be carefully inspected and strictly managed to maintain excellent water quality. The potential risks of algae proliferation in this project should be of concern in future research.