A novel spectrophotometric method based on amino G acid chlorophosphonazo (AGCPA) has been developed for measuring calcium in human serum and cerebrospinal fluids. The effects of experimental factors including pH, reagent concentrations, and interfering species on calcium determinations were investigated. In a neutral reaction media of pH 7.0, AGCPA reacts rapidly with calcium to form a stable and greenish blue complex, whose absorption maximum is at 670 nm. The molar absorptivity (ɛ) of the complex is 7.2x1041 mol−1cm−1, and the molar ratio of calcium to AGCPA in the complex is found to be 1:2. Beer's law is obeyed over the range 0.02-0.8 μg ml−1of calcium. No interferences were observed from the commonly coexisting species present in serum and cerebrospinal fluids. Compared to the reported and currently often used methods based ono-cresolphthalein complexone, arsenazo III and methylthymol blue, the proposed method in this work provides better analytical characteristics such as high sensitivity, good selectivity and neutral reaction media. In addition, the present method is simple and can be applied to the direct determination of calcium in human serum and cerebrospinal fluid samples with satisfactory results.