With the continuous advancement of contemporary medical technology, an increasing number of individuals are inclined towards self-monitoring their physiological health information, specifically focusing on monitoring blood glucose levels. However, as an emerging flexible sensing technique, continuous and non-invasive monitoring of glucose in sweat offers a promising alternative to conventional invasive blood tests for measuring blood glucose levels, reducing the risk of infection associated with blood testing. In this study, we fabricated a flexible and wearable electrochemical enzyme sensor based on a two-dimensional Ti3C2Tx MXene nanosheets and coral-like polyaniline (PANI) binary nanocomposite (denoted as Ti3C2Tx/PANI) for continuous, non-invasive, real-time monitoring of sweat glucose. The exceptional conductivity of Ti3C2Tx MXene nanosheets, in conjunction with the mutual doping effect facilitated by coral-like PANI, significantly enhances electrical conductivity and specific surface areas of Ti3C2Tx/PANI. Consequently, the fabricated sensor exhibits remarkable sensitivity (25.16 μA·mM−1·cm−2), a low detection limit of glucose (26 μM), and an extensive detection range (0.05 mM ~ 1.0 mM) in sweat. Due to the dense coral-like structure of Ti3C2Tx/PANI binary nanocomposite, a larger effective area is obtained to offer more active sites for enzyme immobilization and enhancing enzymatic catalytic activity. Moreover, the sensor demonstrates exceptional mechanical performance, enabling a 60° bend in practical applications, thus satisfying the rigorous demands of human sweat detection applications. The results obtained from continuous 60 min in vitro monitoring of sweat glucose levels demonstrate a robust correlation with the data of blood glucose levels collected by a commercial glucose meter. Furthermore, the fabricated Ti3C2Tx/PANI/GOx sensor demonstrated agreement with HPLC findings regarding the actual concentration of added glucose. This study presents an efficient and practical approach for the development of a highly reliable MXene glucose biosensor, enabling stable and long-term monitoring of glucose levels in human sweat.