As one of the main active components of Dendrobium catenatum, alkaloids have high medicinal value. The physicochemical properties, conserved domains and motifs, phylogenetic analysis, and cis-acting elements of the gene family members in the alkaloid biosynthesis pathway of D. catenatum were analyzed by bioinformatics, and the expression of the genes in different years and tissues was analyzed by qRT-PCR. There are 16 gene families, including 25 genes, in the D. catenatum alkaloid biosynthesis pathway. The analysis of conserved domains and motifs showed that the types, quantities, and orders of domains and motifs were similar among members of the same family, but there were significant differences among families. Phylogenetic analysis indicated that the gene family members showed some evolutionary conservation. Cis-acting element analysis revealed that there were a large number of light-responsive elements and MYB (v-myb avian myeloblastosis viral oncogene homolog)-related elements in these genes. qRT-PCR showed that expressions of gene family members involved in alkaloid synthesis were different in different years and tissues of D. catenatum. This study provides a theoretical basis for further exploration of the regulatory mechanisms of these genes in the alkaloid biosynthesis of D. catenatum.