Penetration ability and accumulation efficiency are two key factors to evaluate the in vivo performance of nanoprobes and demand the different size range, which seems paradox. Herein, we reported the acid-triggered aggregation of mixed-charge ultra-small iron oxide (UIO) nanoprobes to provide a promising solution to meet the size request of different stages in vivo. These nanoprobes were prepared by the coprecipitation of carboxymethyl chitosan (CMC) and iron ions in the hot aqueous acid alkaline solution. For the simultaneously existence of potential negative charge group (-COOH) and positive charge group (-NH2) on the surface of UIO nanoprobes, they displayed the cascade aggregation behavior with the gradually decreased pH value of the surrounding environment. This unique feature of UIO-CMC nanoprobes facilitates them to form the small aggregations to enhance the tumor cell endocytosis in the acidic environment and their tumor tissues accumulation efficiency. This phenomenon enables UIO-CMC nanoprobes selectively enhance MRI signal intensity of tumor tissues 1.86fold that of UIO-CS (chitosan-coated iron oxide nanoparticles) in vivo. Moreover, the modification and evaluation of NBD dye, which enhance the signal intensity in the hydrophobic environment, on the surface of UIO-CMC, not only validate the aggregation behavior of these nanoprobes, but also provide the amplification of fluorescence imaging mode in vitro and in vivo. This study provides a novel paradigm for the design, preparation and application of mixed-charge nanoparticles to selectively enhance the MRI and fluorescence imaging.