l-Asparaginase (l-Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, l-glutamine (l-Gln) and l-asparagine (l-Asn). To study the cytotoxic effect and the secondary complications of l-Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of l-Asnase for the hydrolysis of l-Gln, employing the enzyme-gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)-LIF was established for the separation of l-amino acids (l-Gln and l-glutamic acid) and studying the hydrolysis of l-Gln by using l-Asnase enzyme reactor. Then, using l-Gln as target analyte, the enzyme kinetics of l-Asnase in free solution, enzyme-gold nanoparticle conjugates (E-GNP), and the enzyme-gold nanoparticle conjugates immobilized in capillary (E-GNP-C) were investigated in detail with the proposed MCE-LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of l-Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E-GNP-C enzyme reactor exhibited the best performance for the hydrolysis of l-Gln.