An in-line Mach–Zehnder interferometer (MZI) based on no-core fiber (NCF) with lateral offset for concentration sensing is presented and demonstrated. The in-line MZI is formed by splicing a section of NCF between two single-mode fibers (SMFs) with a lateral offset. The theoretical and simulation analysis results indicate that the interference dip originates from the mode coupling between the high-order mode LP₀₁ and the high-order mode LP₂₆. In the concentration ranges of 0% to 37%, 37% to 66%, and 66% to 82%, the glycerol concentration sensitivities are 0.166, 0.378, and 1.44 nm/%, respectively. The temperature sensitivity is 0.081 nm / ° C from 30°C to 80°C. Our sensor has the advantages such as ease of fabrication, high glycerol concentration sensitivity, and great stability, providing a practical and competitive solution for chemical sensing and food processing.