In order to accurately measure the glucose concentration, the photoacoustic spectroscopy combined with the scanning position method was used in this paper. Meanwhile, a kind of custom-built photoacoustic detection system was established. In this system, a 532nm pumped OPO pulsed laser was used as the excitation source, and a non-focused ultrasonic detector was used to capture the photoacoustic signals of phantom and glucose. To improve the measurement accuracy of glucose by using the photoacoustic spectroscopy, a scanning sub-system was established to accurately locate the position of the simulated vessel. In the experiments, to simulate the bio-tissue and the blood vessel in human body, a specimen made of agar block with a carbon bar and a silicon gel pipe was prepared to verify the availability and feasibility of the established photoacoustic detection system. According to the obtained photoacoustic peak-to-peak values of the specimen by scanning method, the position of the carbon bar and the silicon gel pipe in the specimen can be located. Then, to test the photoacoustic detection of glucose in the silicon gel pipe, a cycling sub-system was used to simulate the blood flow in the blood vessel. Based on the located position of the silica gel pipe, the focused laser spot was shifted and accurately irradiated into the silica gel pipe to induce the photoacoustic signals of glucose. By mean of the photoacoustic system, several different concentrations of glucose solutions were test, and the time-resolved photoacoustic signals and peak-to-peak values of glucose are all obtained. The prediction model of glucose concentration was established by using the linear fitting method. At the same time, the predicted result was compared with that of the glucose in the agar specimen. Results show that the novel photoacoustic detection of glucose is available. The correction coefficient of the glucose concentration prediction was improved by using the photoacoustic spectroscopy combined with the scanning position method.