<p>Photoluminescence imaging is valuable for elucidating biological processes and diagnosing diseases, but its tissue penetration is limited. We developed an imaging technique that utilizes ultrasound to activate the piezoelectric effect of a molecular probe, transforming ultrasound energy into chemical energy. The chemical energy is then converted into light emission through the chemiluminescence effect, improving penetration depth and overcoming traditional photoluminescence imaging constraints. Here we describe how to build two kinds of ultrasound-induced luminescence imaging systems. We introduce a procedure for the synthesis of trianthracene derivative (TD) nanoparticles with ultrasound-induced luminescence properties. The TDs are converted into water-soluble nanoparticles by a simple nanoprecipitation method. Utilizing the constructed ultrasound-induced luminescence imaging systems, TD nanoparticles can be stimulated to exhibit a luminescence spectrum peaking between 625 and 650 nm. Under optimized ultrasound excitation time and excitation power density parameters, the imaging quality and tissue penetration depth are effectively enhanced. Notably, our procedure enables the detection of both subcutaneous tumor models and challenging deep-tissue orthotopic gliomas. This ultrasound-mediated approach represents an important advancement over conventional photoluminescence imaging methods, enabling high-fidelity in vivo tumor imaging with superior signal quality. Establishment of the ultrasound-induced luminescence imaging systems requires ~2 h, the synthesis of TD molecules requires ~4 d, nanoparticle preparation requires ~1 d, ex vivo characterization requires ~1 d, investigation of the ultrasound-induced luminescence of TD nanoparticles requires ~3 d and ultrasound-induced luminescence imaging takes ~1 d. These steps can be performed by operators trained in chemical synthesis, nanomaterial synthesis standards and qualified in relevant animal experiments.</p>