Asthma is a complex disorder, which is known to be affected by interactions between genetic and environmental factors. The aim of this study was to investigate the three microsatellite polymorphisms of GT repeats in intron 2, AAT repeats in intron 20, and CA repeats in exon 29 of the NOS1 gene in 155 asthmatic children and 301 control children, and the interaction with environmental factors in southern Taiwan. Total serum IgE, phadiatop test and genetic polymorphisms were measured. The genotype frequency of 14/14-AAT repeats of the NOS1 gene was significantly higher in the asthmatic group (p = 0.01). Total IgE concentrations were higher in asthmatic children (p = 0.015) carrying the NOS1 14/14-AAT genotype than in subjects with other polymorphisms. The gene and environmental interaction effects were 3.83-fold, 6.86-fold, and 8.04-fold (all corrected p-values <0.001) between subjects carrying at least one NOS1 14-AAT allele and exposure to cockroaches, high levels of total IgE, and positive response against the phadiatop test in asthmatic children. The findings of this study provide strong evidence that NOS1 gene with 14-AAT tandem repeats has a significant effect in asthmatic children. Environmental factors and atopic status will enhance the asthmatic risk for children who carry NOS1 susceptible allele.