Abstract: Cembratrien-diols (CBT-diols) and sucrose esters (SE) are abundant in the glandular trichome secretion of Hongda tabacco flowers. To ascertain the effects of developmental stage and tobacco planting altitude on the glandular trichome secretion in tobacco flowers, Honghuadajinyuan tabacco flowers from Dali were selected to determine CBT-diols and SE by ultra-performance liquid chromatography and gas chromatography mass spectrometry. The results showed that: (1) With the progression of developmental stages, the accumulation of CBT-diols and SE in individual flower and inflorescence increased continuously, and the accumulation in inflorescence reached the highest level at full bloom stage. (2) With the increase of planting altitude, the biological yield of inflorescence evidently decreased, but the CBT-diols and SE levels in individual flower appeared to be increased; the SE level in inflorescence was the highest at high planting altitude (2227 m), yet the level of CBT-diols was significantly lower than that at middle-high planting altitude (1938 m, 1740 m). (3) The ratio of CBT-diols and SE accumulation was significantly influenced by planting altitude and developmental stage, which was the lowest at early tobacco flower development and high planting altitude. The results from this study provide a basis for the utilization of the waste resources of Hongda tabacco flowers with high added value.