The potential toxicity of nanoparticles, especially for clinically applicable ones, has become a critical concern. Technologies that can in situ-evaluate the toxicity of nanoparticles with high sensitivity are urgently needed. In this study, a facile strategy was developed for sensitive detection on the nanotoxicity of nanoparticles with low toxicity or a low dose. A functional nanoprobe loaded with molecular beacons was constructed to realize in situ evaluation of the nanotoxicity through probing multiple miRNAs in nanoparticle-exposed living cells. Being composed of protamine complexed with molecular beacons for miRNA detection and decorated by TAT and KALA peptides, the dual-peptide functionalized nanoprobe can efficiently deliver molecular beacons into living cells to realize the real-time monitoring of early biomarkers (miR-21 and miR-221) to evaluate nanotoxicity. Using mesoporous silica nanoparticles (MSNs) with different surface modifications as typical representatives of low toxic nanoparticles, we demonstrate that our nanoprobe can sensitively detect miRNA changes in cells under diverse exposure conditions, that is, MSN-NH2 exhibits the strongest capability to upregulate miR-21 and miR-221, and the upregulation is exposure dose- and time-dependent. Our approach is much more sensitive as compared with conventional methods to study cytotoxicity such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell morphology observation, and reactive oxygen species (ROS) assay. This study paves a path for effective and facile nanotoxicity evaluation and provides insights into the biological impacts of MSNs.