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The estimation of ambiguity in the global navigation satellite system/inertial navigation system (GNSS/INS) tightly coupled system is a key issue for GNSS/INS precise navigation positioning. Only when the ambiguity is solved correctly can the integrated navigation system obtain high-precision positioning results. Aiming at the problem of ambiguity parameter estimation in GNSS/INS tightly coupled system, a new strategy for ambiguity parameter estimation and elimination is proposed in this paper. Here, the ambiguity parameter is first added to the state equations of GNSS/INS in the estimation process. Then, the strategy of eliminating the parameter is used to improve efficiency. A residual test is carried out based on introducing the ambiguity parameter, thereby reducing or avoiding its influence on the filtering estimation process. Two groups of experiments were carried out and compared with GNSS positioning results. The results showed that, in the open sky observation environment, the positioning accuracy of the GNSS/INS tightly coupled method proposed in this paper was within 5 cm, and the ambiguity fixed rate was more than 97%, which is basically consistent. In a GNSS-denied environment, the positioning accuracy of the GNSS/INS tightly coupled method proposed in this paper was obviously better than that of GNSS, and the positioning accuracy in X, Y, and Z directions was improved by 82.46%, 78.87%, and 79.67%, respectively. The fixed rate of ambiguity increased from 73% to 78.57%. Therefore, in a GNSS-challenged environment, the novel strategy of the GNSS/INS tightly coupled system has higher ambiguity fixed rate and significantly improves positioning accuracy and continuity. [ABSTRACT FROM AUTHOR]