Since China possesses the vast territory and a large blueberry planting area, blueberry harvesting with hand is laborious and time consuming. As the blueberry planting agronomy in China is different from other countries, it is pretty significant to develop blueberry harvesting machinery applicable to the domestic planting agronomy to achieve mechanized harvesting of blueberries. In blueberry harvesting operation, the harvesting system as the core component of the machine is the key technology of the harvesting machinery. The previous study found that: in terms of harvesting machinery, most of the literature has studied the dynamic characteristics, while few articles have been published to study the transmission clearance. But the clearance collision force generated by the transmission clearance of the harvesting system directly affected the output load moment and harvesting force of the machine developed by acting on the plant, and then affected the picking efficiency and the quality of picked fruit. Therefore, this paper focuses on the study of the transmission clearance of the blueberry harvesting machine. Firstly, the MLSD modeling method was applied to establish the transmission clearance model of the harvesting device, and the corresponding mechanical analysis of the transmission clearance was carried out. Secondly, after programming in MATLAB software and simulation in ADAMS software, the correctness of the transmission clearance model of the harvesting device was verified in different environments. The multi-body dynamics analysis software ADAMS was used for building the transmission clearance model of the harvesting device and perform mechanical simulation to analyze the clearance collision force and the output load moment of the harvesting device. Pro/E, ADAMS and ANSYS software were integrated to establish the flexible body of blueberry plant. Then the flexible body was combined with the mechanical model of harvesting device for rigid-flexible coupling simulation analysis to study the fruit harvesting force under different transmission clearances. Finally, the orthogonal method was used to conduct field test on blueberry harvesting to study the influence of the transmission clearance on the quality of picked fruit and picking efficiency of the machinery. Therefore, the best combination of the transmission clearance of the harvester was obtained as follows: cam clearance (the clearance joints of cam) was 0.25 mm, the slider clearance was 0.2 mm, left connecting rod clearance pair was 0.1 mm, right connecting rod clearance pair was 0.2 mm; the field picking test was conducted to obtain the machine's picking efficiency was 3.93 kg min−1, the shedding rate of unripe fruit was 3.1 %, and the damage rate of picked fruit was 2.8 %. The research findings of this paper can offer referenced basis and theoretical support for berry harvesting machinery, and also can provide guidance for the design and improvement of other agricultural and forestry harvesting machinery.