A flow injection analysis (FIA) method was developed for the rapid and environmentally friendly determination of the acidity of thermal conductive oil. The acid number (AN) of the oil sample was obtained by online measurement of the concentration change of the standard KOH solution before and after the interaction with the oil sample. To ensure the adequate reaction of KOH with the acid in oil, the oil sample and KOH solution were segmented into small droplets in the confluence tube with the help of a coiled wire. The parameters, such as the mixing ratio of KOH with oil, the length of mixing tube, and the flow rate of the two mixing streams, were optimized. Then, the FIA manifolds was integrated to a three-dimensional (3D)-printed FIA system (3DFIA), which was used for the determination of the AN in real oil samples. The results showed that the AN values in the range of 0.206.4 mg of KOH/g oil can be accurately determined with a limit of detection of 0.177 mg of KOH/g and a sampling frequency of 10 h1. Also, the results obtained with the 3DFIA system agreed well with that obtained from the conventional FIA system as well as the standard titration method. The new method not only exhibited good precision and rapidity of analysis but also completely eliminated the use of organic solvents.