Litter humification plays a crucial role in organic matter formation and soil carbon sequestration in forest ecosystems. However, how forest gap formation and gap size variation affect the litter humification process remains poorly understood. An eight-year in situ decomposition experiment was conducted to evaluate humus accumulation (humic substances, humic and fulvic acid), humification degrees, humification ratios and optical properties (ΔlogK, E4/E6 and A600/C) of Minjiang fir (Abies faxoniana Rehder & E.H.Wilson) twig litter in four gap size treatments in an alpine primitive forest on the eastern Tibetan Plateau, including (1) closed canopies, (2) small gaps (38–46 m2 in size), (3) medium gaps (153–176 m2 in size),and (4) large gaps (255–290 m2 in size). The results indicated that the accumulation of humic substances and humic acid in the closed canopies was significantly higher than that in the large gaps during the first two years of decomposition. After eight years of decomposition, there were significant differences in the humic substance accumulations and the values of ΔlogK and A600/C among the different gap sizes. Furthermore, twig litter was humified in the first 2 years of incubation, and the net accumulation of humic substances was ranged from −23.46% to −44.04% of the initial level at the end of the experiment. The newly accumulated humus was young (mature (type Rp) humus) and transformed to mature (type A) humus after 4–6 years of decomposition. Partial least squares (PLS) suggested that gap-induced variations in twig litter chemistry (i.e., contents of cellulose, lignin, nitrogen (N) and phosphorus (P), and the ratios of C/N N/P) mainly drove the process of twig litter humification. Our results presented here denote that the formation of forest gaps retard twig litter humification process, which might be detrimental to carbon sequestration in the alpine forest ecosystems.