Much research has been carried out on nanoscale polymer microspheres (PMs) in laboratories in recent years. However, there are limited reports on the practical application of nanoscale PMs in ultralow permeability reservoirs. This paper reports a field application case of nanoscale PMs for in-depth profile control in the ultralow permeability oil reservoir. In the paper, the characteristics of the reservoir and the problems faced during development are analyzed in detail. Then, the PMs with calibration diameters of 300 nm and 800 nm are researched by evaluation experiments, and are selected for in-depth profile control in the ultralow permeability oil reservoir. Finally, according to the effect of the pilot application, the performance of PMs is evaluated, and a more suitable size for the pilot test reservoir is determined. The experiment's results show that the PMs have a good capacity for swelling and plugging. For the PMs with a calibration diameter of 300 nm, the final equilibrium swelling ratio is 56.2 nm·nm
−1
, and the maximum resistance coefficient and the blocking rate after swelling are 3.7 and 70.31%, respectively. For the PMs with a calibration diameter of 800 nm, the final equilibrium swelling ratio is 49.4 nm·nm
−1
, and the maximum resistance coefficient the blocking rate after swelling are 3.5 and 71.42%, respectively. The performance evaluation results show that nanoscale PMs can be used for in-depth profile control in the ultralow permeability oil reservoir. After the application of PMs in the pilot test area, the average water cut decreased by 10.4%, the average liquid production of single well-increased by 0.9 t/d, and the average thickness of the water-absorbing layer increased by 1.77 m. Comparing the dynamic data variation of well-groups using the PMs with the calibration diameter as 800 nm and the calibration diameter as 300 nm, it indicates that, for the pilot test area, PMs with a calibration diameter of 300 nm are more suitable than PMs with a calibration diameter of 800 nm.