Background: An increasing number of studies have explored disease and growth traits through quantitative trait locus (QTLs) in chickens. Nevertheless, the pathogenic genes underlying the QTL effects remain poorly understood. Alkaline phosphatase (ALP) is a marker of osteoblast maturation and an important indicator of bone metabolism. The change of ALP can reflect the bone metabolism and growth traits of animals to a certain extent. Results: In this study, we identified a SNP site at ST3GAL4 that found by genome-wide association studies (GWAS) in previous studies, and found another 8 SNPs by DNA sequencing. Interestingly, there are 4 SNPs rs475471G>A, rs475533C>T, rs475621A>G, rs475647C>A were completely linked by haplotype analysis, and selected a tag SNP rs475471G>A to analyze the correlation between this SNP and ALP level in Hubbard leg disease population and an F2 chicken resource population produced by Anka and Gushi chickens, and carried out population genetic analysis in 18 chicken breeds. Association analysis showed that this QTL within ST3GAL4 were highly correlated with ALP level, the mutant individuals with genotype AA had the highest ALP level, followed by GA and GG genotypes. The mutant individuals with genotype AA and GA genotype had higher values for body weight (BW), chest width (CW), body slanting length (BSL), pelvis width (PW) at 4-week, the semi-evisceration weight (SEW), evisceration weight (EW) and Leg weight (LW) of AA and GA genotype also higher. The amplification and typing of 4,852 DNA samples from 18 different breeds and the result shown GG genotype mainly existed in egg-type chickens and dual-type chickens, while AA genotype mainly distributed in commercial broilers and F2 resource population. The individuals of AA genotype had the highest ALP and showed better growth performance. This is the first time to report the causal variant of ST3GAL4 gene that located at chromosome 24 related to chicken serum ALP level. Besides, tissue expression analysis used Cobb broiler showed that there were significant differences between different genotypes in spleen and duodenum.Conclusions: This study the first time to determine 9 SNPs within ST3GAL4 were related to ALP in chickens, 4 of them were complete linkage sequence variants, which provide useful information on the mutation of ST3GAL4 and could predict the serum ALP level of chicken early and effectively as a potential molecular breeding marker.