Cell identification is an important yet difficult process in data analysis of biological images. Previously, we developed an automated cell identification method called CRF_ID and demonstrated its high performance inC. eleganswhole-brain images (Chaudhary et al, 2021). However, because the method was optimized for whole-brain imaging, comparable performance could not be guaranteed for application in commonly usedC. elegansmulti-cell images that display a subpopulation of cells. Here, we present an advance CRF_ID 2.0 that expands the generalizability of the method to multi-cell imaging beyond whole-brain imaging. To illustrate the application of the advance, we show the characterization of CRF_ID 2.0 in multi-cell imaging and cell-specific gene expression analysis inC. elegans. This work demonstrates that high accuracy automated cell annotation in multi-cell imaging can expedite cell identification and reduce its subjectivity inC. elegansand potentially other biological images of various origins.