This study is intended to develop an assessment instrument to investigate students' understandings about internal transport in plants and human circulatory system. A refined process of a two-tier diagnostic test was used to develop the instrument. Finally, three versions of the Internal Transport in Plants and the Human Circulatory System test were developed for different educational levels: elementary, secondary and high school level. The test-retest reliability, the degree of difficulty, and the range of discrimination were found to be reasonable for all three versions. The statistical variances across educational levels of the verification samples and the test applications are discussed. The students' major alternative conceptions on internal transport of plants and human were classified into three attributes of explanations: interconnectedness, language and anthropomorphism. Based on the nature of students' explanations, card-sorting with justification, learning cycle and concept mapping were recommended for in-depth learning of the concept.