The CDS region of ADD1 gene of Guangling donkey was cloned by RT-PCR method, and followed with sequence analysis. qRT-PCR was used to identify the relative expression level of ADD1 gene in heart, liver, spleen, lung, kidney and longissimus dorsi muscle. The results showed that the complete CDS sequence of Guangling donkey ADD1 gene was 2 223 bp in length and could encode 740 amino acids. The sequence was submitted to NCBI with the login number MN_166472, and its nucleotide sequence had the highest homology with the horse nucleotide sequence, up to 99.6%. Bioinformatics prediction ADD1 protein had stable hydrophilic protein structure, theory of isoelectric point was 5.58, the secondary structure was mainly α-helix and random coils, and there was a Aldolase_Ⅱsuperfamily domain in the protein sequence. The protein had no signal peptide and transmembrane region, and was mainly located in the cell nucleus. There were 88 phosphorylation sites, 69 O-glycosylation sites and 3 N-glycosylation sites in the sequence. The results of real-time fluorescence quantitative detection showed that ADD1 gene was expressed in all studied 6 tissues, but there were differences in expression. The expression of ADD1 gene was the most abundant in longissimus dorsi muscle and the lowest in liver.