Atherosclerosis is closely related to various cardiovascular diseases, directly leading to arterial wall hardening, thereby producing a series of vascular physiological function abnormalities and inducing various cardiovascular diseases. Therefore, the detection of atherosclerosis is crucial in cardiovascular medicine. In order to verify the photoacoustic platform’s ability to detect atherosclerosis in living tissues and explore the application prospects of photoacoustic spectrum analysis in peripheral blood vessel detection, we carried out simulation and in vivo animal experiments. The K-wave simulation verified the theoretical feasibility of photoacoustic spectrum analysis. A photoacoustic detection system was developed, and an atherosclerosis mouse model fed with a high-fat diet was established for collecting the corresponding signal data and performing analysis. The spectral data of the photoacoustic signal of atherosclerosis model mice and normal mice are significantly separated after variance analysis, and the spectrum analysis results of the experimental data are in good agreement with the K-wave theoretical analysis results. Experimental results show that photoacoustic spectrum analysis shows potential for detecting arteriolosclerosis in vivo. This analysis is expected to be an effective method for detecting arteriolosclerosis.