This study aimed to investigate the variations in the content of small molecule metabolites in two-year-old Dendrobium officinale at different harvesting periods, with the goal of determining the optimal harvesting time to obtain the desired metabolite content. Gas chromatography-mass spectrometry and ultra-performance liquid chromatography-mass spectrometry were employed to conduct an in-depth analysis of three categories of small molecule metabolites: sugars, alkaloids, and amino acids. The results revealed that the accumulation trends of glucose, fructose, and sucrose, which were present in higher concentrations, initially increased and then decreased from mid-November to early April of the following year, peaking between January and March, before beginning to decline in mid-March. Among the alkaloids, choline, dendrobine, and isodendrobine were the most abundant, accounting for 89.20%, 88.05%, 90.11%, and 92.80% of the total relative alkaloid content across the different harvesting periods, respectively. Differential analysis indicated that the relative content of each alkaloid varied across the harvesting periods. The total content of 18 amino acids in two-year-old D. officinale also varied, with the highest content (0.484%) observed in early April of the following year and the lowest (0.09%) in mid-November. Based on the comprehensive analysis of the three categories of small molecule metabolites, it is recommended to harvest two-year-old D. officinale in mid-February, mid-November, or early April of the following year to obtain higher concentrations of sugars, alkaloids, and amino acids.