AbstractAcute cardiac allograft rejection (ACAR) is one of the main reasons for mortality after heart transplantation. The timely diagnosis of treatment‐needed ACAR is of great importance in the clinic. Molecular imaging may make accurate diagnoses at the early stage to visualize the activity of specific pathological processes. The amount of CD4+‐lymphocyte infiltration is a crucial index in ACAR grading. CD4‐targeted nanobubbles (NBCD4) are fabricated to assess the infiltrating CD4+ lymphocytes, while isotype control nanobubbles (NBIso) are also fabricated. Animal models with ACAR grades from 0R to 3R are established. Ultrasound molecular imaging (USMI) is performed to distinguish different rejection grades with NBCD4. In the grade 2R and 3R groups, the USMI signals of NBCD4 are significantly higher than those of NBIso. The signals of NBCD4 in the 2R group are significantly lower than those in the 3R group and significantly higher than those in the 1R and 0R groups. Moreover, the signals of NBCD4 are strongly correlated with the rejection grades and the number of infiltrating CD4+ lymphocytes. As asymptomatic rejection with 2R or higher grades should be seriously treated, USMI with NBCD4 may be a new approach to timely diagnosis of treatment‐needed ACAR.