Dendrobium officinale is a medicinal orchid renowned for its rich polysaccharide content and significant health benefits, such as nourishing Yin, moistening the lungs, and enhancing immunity. Pectin methylesterase (PME), a key enzyme catalyzing pectin demethylation, plays critical roles in plant cell wall modification, growth, development, and stress responses. The activity of PME is finely regulated by pectin methylesterase inhibitors (PMEIs). Investigating the characteristics and functions of the PME gene family in D. officinale is essential for improving its medicinal value and stress resilience. This study based on chromosome-level genome data of D. officinale,systematically identified the PME and PMEI gene family in D. officinale using a combined approach, revealing 46 DoPME (33 were classified as type I and 13 as type II) and 31 DoPMEI genes. Phylogenetic analysis grouped DoPMEs into four clades (A–D) and DoPMEIs into three clades (A–C). Conservation within clusters was supported by analyses of domains, motifs, and gene structures. Syntery analysis indicated that the evolutionary trajectory of these gene families aligns with early divergence followed by prolonged independent evolution in orchids, culminating in stability among closely related species. Expression profiling via qRT-PCR demonstrated tissue- and developmental stage-specific patterns, with the highest expression levels observed in roots, flowers, and perennial stems. This study provides the first comprehensive analysis of the DoPME and DoPMEI gene in D. officinale, offering foundational insights into their biological functions and potential genetic resources for enhancing stress tolerance and medicinal quality.