In this work, a novel ratiometric fluorescent nanosensor by coupling mesoporous silica spheres-coated nitrogen-doped carbon dots (N-CDs@SiO) and bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) was developed for sensitive detection of glyphosate. BSA-AuNCs nanocomposites were covalently connected onto the surface of amino-functionalized N-CDs@SiO nanospheres, causing the core-satellite probes with two fluorescence emissions at 436 and 651 nm, respectively, under a single excitation of 360 nm. The N-CDs@SiO@BSA-AuNCs “signal on-off-on” ratiometric fluorescent probes could realize highly selective differentiation and accurate detection of glyphosate based on the fluorescence quenching of Cu on BSA-AuNCs and glyphosate-induced fluorescence recovery due to the strong complexation of glyphosate toward Cu. The developed ratiometric fluorescent nanosensor exhibited excellent selectivity to glyphosate, wide detectability in a concentration range of 5-100 ng/mL, high sensitivity with a low detection limit of 3.4 ng/mL, as well as fascinating reliability and practicability in real malt samples with recoveries of 94.81-101.61%. This study provides a universal platform for versatile sensing of more trace analytes in complex matrices and can be extended for wide application in the environment and food safety fields.