In this paper, a low-cost, rapid, automated centrifugal microfluidic chip for biochemical analysis was presented. The chip integrates sample pre-processing and multi-biochemical items assay. Through multi-level microfluidic channels and micro-siphon valves, the chip combines the pre-treatment of samples, sample transportation and the reaction processes between sample and biochemical reagent. The results are obtained by colorimetric detection. It is shown that the reproducibility of cuvette pathlength for chip is from 0.08% to 0.52%. The coefficient of variation (CV) of sample, diluents metering and mixing for chip by the CV of blood glucose results is 1.4%. The CV of day-to-day and within-run precision for 15 biochemical items are less than 3.5%. Therefore, it is concluded that the centrifugal microfluidic chip for biochemical analysis can satisfy the clinical testing requirements.