The spiritual (S) aspect needs to be integrated with the environmental literacy instrument (ELI) sothat it becomes ELIS. This study was designed to develop and validate an instrument forprospective science teacher in the form of environmental literacy instrument based on literacy(ELIS). The instrument was developed based on previous research findings and focus groupdiscussions involving eleven experts. The factors and internal consistency were examined byinvolving 634 students/prospective science teachers from various universities in Indonesia.Validity and reliability were tested using exploratory factor analysis and confirmatory factoranalysis. This study proposes a 26-item ELIS with acceptable internal validity and consistency. It iscomposed of four dimensions: ecological knowledge (five items), environmental hope (sevenitems), cognitive skills (eight items), and behavior (six items). The ELIS is recommended as ameasure of the development of environmental literacy based on spirituality for prospectivescience teachers.