A simple and sensitive spectrophotometric method, based on reaction between Ni(II) ion and D-penicillamine (PEN), was developed. The proposed SIA system enhanced the analytical applicability of the reaction of complexation, and allowed the determination of PEN in the concentration range of 3.0 × 10(-6) - 2.0 × 10(-4) mol L(-1) with a sampling rate of 200 h(-1). With the proposed SIA system, PEN could be accurately determinated up to 0.9 nmol quantity. The method was successfully applied to the determination of PEN in laboratory samples and pharmaceuticals.