Harvest aids are widely used for defoliating leaves and accelerating the opening of green bolls to facilitate machine harvesting in cotton (Gossypium hirsutum L.) production areas. Cotton harvest aids applied by ground-based mechanical sprayers are inefficient due to mechanical damage to cotton crops and soil and low flexibility. For the last few years, small plant protection unmanned aerial vehicles (UAVs) have been used for applying pesticides across the world due to their high efficiency, high pesticide utilization, low volume and no harmful damage to crops and soil. This study mainly focuses on developing the technology of harvest aid application by UAVs with respect to the dosage and application frequency. Compared with previous studies, this work performs miscellaneous field trials for two years in three experimental sites located in high-density planting areas and two sites in sparse-density planting areas, wherein both cotton cultivation modes and weather conditions are different. In the study, single-round, dual-round and reduced dosage applications are tested, where the defoliation rate, boll opening rate, fiber quality and lint cotton yield are assessed based on the collected data. It is concluded from the experimental results that the achieved defoliation rate and boll opening rate of treatments with a single-round application using the recommended dosage fail to meet the harvest requirements in the case of high planting density (180,000-195,000 plants/hm 2). However, with the dual-round application of the exact recommended dosage or 20% lower than the recommended dosage, the achieved defoliation rate, and boll opening rate meet the machine harvest requirements. In sparse-density planting areas (≤90,000 plants/ha), the results of treatment with the recommended dosage and single-round application by UAV spraying meet the requirements. In all the experimental sites, the harvest-aid dosage and application frequency do not affect fiber quality and lint cotton yield. In summary, considering the cost and environmental protection, harvest aid application by UAVs with a dual-round application at 80% of the recommended dosage at a 7-day interval is encouraged in high-density planting areas, while in sparse-density planting areas, single-round application of harvest aids at the recommended dosage by UAVs is encouraged. The results provide paramount guidance for cotton farmers and scholars in this field. Possible future studies are also discussed in this paper.