Abstract: In order to establish the flow cytometry (FCM) method for determination of macadamia genomic C value, The tender leaves of macadamia cultivar ‘O.C’ was used as material, the adjusted OTTO and PI were used as isolation buffer and DNA fluorochrome, the Pisum sativum L. Ctirad and Zea mays L. CE-777 were used as DNA reference standards, the genomic C value was determined by FCM. The result showed that the DNA content of diploid macadamia spp. was 1.91488599 pg and the genomic C value was 0.957443 pg; the genome size of macadamia spp. was 1.872758×109 bp calculated by 1pgDNA=0.978×109 bp. The results determined by two different DNA reference standards have no significant difference. The study found that the FCM method was accurate and reliable, it provided a valuable reference for genomic C value determination of other species.