Background <p>Chimeric antigen receptor (CAR) T cells have long been regarded as living drugs, those activation, diffusion, and expansion influence the clinical efficacy. Dynamic monitoring of CAR-T cells in vivo is crucial for understanding their biodistribution and optimizing therapeutic outcomes. However, due to the challenges in sampling tissue-resident CAR-T cells from patients, limited data have been reported on their spatial and temporal distribution.</p> Methods <p>This study enrolled 43 patients with haematological malignancies receiving CAR-T cell therapy. CAR copy numbers in the peripheral blood (PB) and serous cavity effusion (SCE) were sequentially quantified and analysed.</p> Results <p>High expansion of CAR-T cells in PB was associated with subsequent expansion in SCE. The median T_max in SCE occurred later than in PB. Patients with immune effector cell-associated neurotoxicity syndrome (ICANS) exhibited higher CAR copy numbers in cerebrospinal fluid (CSF) compared to those without ICANS. Peripheral infection was associated with increased CAR copy numbers in CSF, which may be caused by cell diffusion or/and expansion. Tumour invasion favored local accumulation and expansion of CAR-T cells in pleural effusion or ascites (PE/A), and patients with tumour invasion had a higher incidence of local cytokine release syndrome (L-CRS).</p> Conclusion <p>We characterized the spatial and temporal distribution of CAR-T cells and identified associations between CAR copy numbers and local inflammation, tumour invasion, and adverse events. These findings enhance our understanding of CAR-T cells diffusion, trafficking, and expansion, providing novel insights for clinical management and therapeutic optimization.</p>