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Objectives: BeiDou-3 navigation satellite system (BDS-3) has been fully completed and pro‐ vides reliable positioning, navigation, and timing services to global users. In order to achieve compatibility and interoperability with other global navigation satellite systems (GNSS), B1C and B2a signals have been modulated by BDS-3 based on BDS-2, which enable frequency multiplexing with E1 and E5a signals of Galileo. The inter-system biases (ISBs) are important for the fusion processing of different GNSS. Methods: This paper proposes an ISB estimation and application model based on the single-differenced observations and analyzes the ISBs between the overlapping frequencies of BDS-3 and Galileo. Based on several receivers of the new trackable BDS-3 signals, the characteristics of ISBs between BDS-3 and Galileo are revealed, and the real-time kinematic (RTK) positioning performance of BDS-3 and Galileo is analyzed. Results: The results show that the ISBs of B1C-E1 and B2a-E5a exist in different types of receivers but not in the same type of receivers. In addition, compared with BDS-3-only and Galileo-only, the combination of BDS-3 and Galileo has an improvement of more than 10% in both the success rate of ambiguity resolution and positioning accuracy. Conclusions: The multi-constellation RTK positioning performance with the introduction of ISBs is more advantageous than that of a single constellation [ABSTRACT FROM AUTHOR]