Somatic embryogenesis is one mode of reproduction in plants, whereas ex vivo callus embryogenesis can perform plant regeneration, but also can be employed for genetic transformation. This study employed the widely targeted metabolomics and explored respectively proliferating and differentiating calli in the fern Ceratopteris richardii, aiming to investigate metabolic mechanisms for callus embryogenesis and thus providing materials for metabonomics of callus embryogenesis. The results found: (1) both the proliferating and the differentiating callus possessed the same metabolites, and there were some disease drugs among them such as actidione and 3［(Carboxycarbonyl)amino］Lalanine. (2) Each metabolites had different contents, but basically they were not altered between the proliferative and the differentiating stages. (3) Metabolites accumulated in the proliferating stage were predicted related to the ABC transporter protein pathway, while those in the differentiating stage probably concerning the flavonoid biosynthesis pathway. The research speculated transition from proliferating to differentiating phase of calli in C. richardii was a quantitative rather than a qualitative process in terms of metabolite alterations, and metabolomes of plant callus embryogenesis might be characterized by species specificity.