To achieve foreign protein expression in the chloroplast of Phaeodactylum tricornutum, a chloroplast transformation vector was constructed. The sequences rns-trnI and trnA-rnl from P. tricornutum chloroplast genome were cloned and used as homologous recombination elements. Chloramphenicol acetyltransferase gene (CAT) expression cassette conferring chloramphenicol resistance was employed as selection marker, and green fluorescent protein gene (GFP) as reporter gene. Based on the TA cloning vector pMD19-T, CAT and GFP expression cassette were cloned in between the two homologous recombination elements. The resultant chloroplast transformation vector was transferred into the chloroplast of P. tricornutum by electroporation method. Transplastomic P. tricornutum cells were obtained upon chloramphenicol selection and reporter GFP could be detected in the chloroplasts, indicating that chloroplast expression system was successfully achieved.