Provided are a near-infrared II polymer fluorescent sub-microsphere and a method for preparing the same. The method includes steps of 1) dissolving fluorochrome in a water-immiscible organic solvent, thus obtaining a fluorochrome solution; 2) distributing a polymer sub-microsphere into a sodium dodecyl sulfonate solution, thus obtaining a sub-microsphere solution with the polymer sub-microsphere as a carrier for the fluorochrome; 3) subjecting a first mixture of the fluorochrome solution and the sub-microsphere solution to ultrasonic treatment, thus obtaining an emulsion; 4) swelling the emulsion such that the fluorochrome solution enters nanopores formed during swelling of the polymer sub-microsphere, thus obtaining a second mixture; and 5) heating the second mixture to volatilize the organic solvent, such that the fluorochrome is crystallized out and encapsulated in the nanopores, thus obtaining the near-infrared II polymer fluorescent sub-microsphere.