The invention discloses an isolated culture and rapid propagation method of Ilex asprella. The method includes the steps: replacing CaCl2*2H2O in an MS medium by Ca(NO3)2*4H2O, adding ferrous sulfate,sucrose and agar to prepare an improved MS medium, and inoculating an explant of the Ilex asprella into the improved MS medium added with 6-BA and IBA to perform inducing culture; inoculating taken adventitious buds of the Ilex asprella into a cluster bud proliferation medium based on the improved MS medium and added with 6-BA, AD and NAA, transferring the adventitious buds to a rooting medium, and adjusting the dosage of a lot of elements in the improved MS medium as half of the original dosage, and adding activated carbon, NAA and IBA; finally, performing indoor acclimatization and transplantation on Ilex asprella seedlings to obtain the Ilex asprella. According to a tissue culture technique, a lot of Ilex asprella seedlings are supplied in a short term, the problem of progeny segregation is solved, a supply market is guaranteed, incomes of planters and sellers are improved, and comprehensive utilization of the Ilex asprella is facilitated.