Fatty acids are important components of the avocado mesocarp, so a better understanding of how their change during fruit development will contribute to improving the quality of avocado fruits and their nutritional value. The changes in fatty acids, lipid droplets, and expression of some key genes and regulators participating in late glycolysis and fatty acid biosynthesis were analyzed at different stages of the development of avocado mesocarp. The total fatty acid contents of the avocado mesocarp increased during fruit development, with an increase by a factor of seven (from 1,628.04 to 11,116.30 mg/100 g dry weight) in the late stage of fruit maturation, this was confirmed by the changes observed in the lipid droplets. The composition of the main fatty acids varied at four developmental stages of fruit development. Palmitic, palmitoleic, oleic, and linoleic acid contents generally increased during fruit development, reaching maxima at Harvest, with percentages of total fatty acids of 50%, 9%, 31%, and 8%, respectively. Meanwhile, the amount of PaWRI1, PaACP4-2, and PapPK-β1 expressed consistently increased by up to 4-fold during fruit development. This comprehensive analysis has indicated that the changes in the expressions of PaWRI1, PaACP4-2, and PapPK-β1 were consistent with those in the total fatty acid contents, so they might have key roles in the accumulation of oil in the avocado mesocarp.