The roots of Paeoniae lactiflora Pall. are widely consumed as crude drugs in Asian countries due to their remarkable beneficial health effects. The present research was undertaken to illuminate the dynamic changes in metabolites and enzymes and facilitate selection of the harvesting time when the herb can provide optimum health benefits. P. lactiflora roots were analyzed at 12 stages of growth for monoterpenoid glycosides, phenols, nucleosides, nucleobases, amino acids, and polysaccharides by high-performance liquid chromatography with photodiode array detector, ultra-high pressure liquid chromatography coupled with tandem mass spectrometry, and UV spectrophotometry. The enzyme activities of plant β-glucosidases and esterases were determined by UV methods. The total content of monoterpenoid glycosides and phenols peaked in December. For nucleosides and nucleobases, the highest content appeared in April. The maximum phasic accumulation of the total amino acids took place in March, and the content of total polysaccharides reached a peak value in September. December, April, and March were selected as the appropriate harvesting times for producing natural medicinal or health food products. Plant β-glucosidases and esterases showed the highest activity in December and May, respectively. When the activity of β-glucosidase increased, esterase activity decreased, while the contents of oxypaeoniflora and paeoniflorin increased. When esterase activity increased, the contents of benzoylpaeoniflorin, paeoniflorin, and gallic acid decreased. In conclusion, the results from the present study would be useful in determination of the suitable time for harvesting P. lactiflora roots for medicinal purposes.