Effective acquisition of transition probability matrix is directly related to Internetware reliability computation. The characteristics of Markov chain in Internetware are discussed and analyzed, the construction of Markov chain and the acquisition of transition probability of Internetware are studied, the Internetware model based on Markov chain is constructed. Quantitative calculation method of transition probability based on the smallest quadratic difference is presented by using the occupancy of component executing the transition as the sample statistics to calculate transition probability. The approximation algorithm for computing transition probability matrix based on the modified projection gradient is designed, and it effectively guarantees the transition law of Markov chain and the characteristics of transition probability matrix. The experiment proves that the presented method and the designed algorithm can effectively compute transition probability matrix with great value in Internetware reliability computation.