We describe a novel multiplex "amino acid array" for simultaneously quantifying different amino acids based on the rapid growth of amino acid auxotrophic E. coli. First, we constructed genetically engineered amino acid auxotrophs of E. coli containing a bioluminescence reporter gene, yielding concomitant luminescence as a response to cell growth, and then immobilized the reporter cells within individual agarose of respective wells in a 96-well plate serving as a mimic of a biochip. Using the amino acid array, we were able to determine quantitatively the concentrations of 16 amino acids in biological fluid by simply measuring bioluminescent signals from the immobilized cells within 4 h without pre- and post-treatment. The clinical utility of this method was verified by quantifying different amino acids in dried blood spot specimens from clinical samples for the diagnosis of metabolic diseases of newborn babies. This method serves as a convenient route to the rapid and simultaneous analysis of multiple amino acids from complex biological fluids and represents a new analytical paradigm that can replace conventional, yet laborious methods currently in use.