Activatable afterglow luminescence nanoprobes enabling switched "off-on" signals in response to biomarkers have recently emerged to achieve reduced unspecific signals and improved imaging fidelity. However, such nanoprobes always use a biomarker-interrupted energy transfer to obtain an activatable signal, which necessitates a strict distance requisition between a donor and an acceptor moiety (<10 nm) and hence induces low efficiency and non-feasibility. Herein, we report organic upconversion afterglow luminescence cocktail nanoparticles (ALCNs) that instead utilize acidity-manipulated singlet oxygen (O) transfer between a donor and an acceptor moiety with enlarged distance and thus possess more efficiency and flexibility to achieve an activatable afterglow signal. After in vitro validation of acidity-activated afterglow luminescence, ALCNs achieve in vivo imaging of 4T1-xenograft subcutaneous tumors in female mice and orthotopic liver tumors in male mice with a high signal-to-noise ratio (SNR). As a representative targeting trial, Bio-ALCNs with biotin modification prove the enhanced targeting ability, sensitivity, and specificity for pulmonary metastasis and subcutaneous tumor imaging via systemic administration of nanoparticles in female mice, which also implies the potential broad utility of ALCNs for tumor imaging with diverse design flexibility. Therefore, this study provides an innovative and general approach for activatable afterglow imaging with better imaging performance than fluorescence imaging.