A sequential injection analysis (SIA) system based on polydimethylsiloxane (PDMS) chip with integrated pneumatic-actuated valves was developed. A novel SIA operation mode using multiphase laminar flow effect and pneumatic microvalve control was proposed. The sample and reagent solutions were synchronously loaded and injected in the chip-based sample injection module instead of multi-step sequential injection by a multiposition valve and a reciprocating pump as in conventional SIA system. The sample and reagent injection volumes were reduced to ca. 1.1 nL. The present system has the advantages of simple structure, fast and convenient operation, low sample and reagent consumption, and high degree of integration and automation. The system operation conditions were optimized using fluorescein as model sample. Its feasibility in biological analysis was preliminarily demonstrated in enzyme inhibition assay. A sequential SIA operation mode using multiphase laminar flow effect and pneumatic microvalve control was proposed. The sample and reagent solutions were synchronously loaded and injected in the chip-based sample injection module instead of multi-step sequential injection by a multiposition valve and a reciprocating pump as conventional SIA system. The sample and reagent injection volumes were reduced to ca. 1.1 nL. The present system has the advantages of simple structure, fast and convenient operation, low sample and reagent consumption, and high degree of integration and automation. The system operation conditions were optimized using fluorescein as model sample. Its feasibility in biological analysis was preliminarily demonstrated in enzyme inhibition assay.