This research aims to describe the characteristics of biology test items in the test instrument of USBN (National Standard School Examination) through test items analysis to check of reliability estimation, difficulty level, and item discrimination, as well as to identify items that have a high level of difficulty, and to analyze the factors that cause the items these questions are difficult for students. Data collection technique was done through documentation. The collected data were in the form of a set of Biology National Standard School Examination key answers, and students' response of senior high school in Banjarbaru City, South Kalimantan Indonesia. Quantitative data analysis was conducted by using the approaches of Classical Test Theory (CTT) and Item Response Theory (IRT). Qualitative data analysis was carried out to describe students' difficulty in answering test items with difficult category. The results show that reliability estimation is quite reliable. Based on the analysis result of CTT and IRT, the mean of difficulty level is categorized as medium, and the mean of distinguishing ability is categorized good. In qualitative analysis, it is obtained that one item becomes difficult to answer due to the low concept comprehension, unfinished learning materials, and learning difficulties during the class.