Penicillin sodium is a commonly used clinical antibiotic. It is noted that the dry powder is stable, but in water solution it is very unstable. It is easily decomposed and destructed by acid, alkali, alcohol, oxidative agent, metal ion. Thus, to ensure the efficiency of penicillin solution, it is clinically required to prepare it just before use. In some cases, it is necessary to timely detect the concentration changing. There are many methods to check the content of the penicillin solution, such as, the iodometric method, pH fixed titration, spectrophotometric method, HPLC method, and optical method. In the literature a linear relationship between optical rotation and concentration at lower concentration of 1.0 mg/ml - 10.0mg/ml has previously been reported. In this paper, we investigated content determination of penicillin at higher concentrations based on polarimetric method, and studied the role of the influencing factors. The experimental results showed that penicillin solution at 10.0 mg/ml - 30.0 mg/ml high concentration also show a good linear relationship, the linear equation is y = 0.2903x + 0.1712, the correlation coefficient r = 0.9993. In contrast with the Pharmacopoeia of iodine quantity method, optical method is simpler, more rapid, more accurate, more reliable, and it can save experimental materials. Moreover, to a certain extent, it can eliminate the influence of other factors. It is a very suitable method for general medical institutes to rapidly detect the content of penicillin solution.