Ichthyophthirius multifiliis is the causative agent of “white spot” disease, which affects numerous freshwater fish and causes severe economic losses to worldwide aquaculture. The disease of I. multifiliis infection could develop very fast, and there are few practical measures to treat this heavy infection. To overcome the shortcomings of existing methods, for detecting I. multifiliis in the early stage and environmental samples, such as low sensitivity of visual diagnosis with microscope and low specificity of PCR methods developed in the previous studies, here we developed and validated novel PCR assays. In this study, a pair of primers (qIchF 5′-TTCTGCCCGTACTTTAGTTACC-3′ and qIchR 5′-TGGTTGTACTAACACCTGCAA-3′) were designed and screened to target the mitochondrial COⅠ gene of I. multifiliis. The length of the amplified product is 131 bp. After PCR programme optimization, specificity and sensitivity verification, clinical and environmental samples detection and analysis, standard PCR and real-time PCR assays were established, respectively. The standard PCR was incubated at 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 15 s, annealing at 51 °C for 15 s, and extension at 72 °C for 10 s; and final extension at 72 °C for 5 min. For real-time PCR was incubated at 95 °C for 30 s; 40 cycles of denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s. The results showed that the primers obtained in this study had high amplification specificity for I. multifiliis. Ciliates Paramecium sp., Tetrahymena sp., and Balantidium sp. were not amplified, nor were common farmed fish hosts Carassius auratus gibelio, grass carp (Ctenopharyngodon idella), Nile tilapia (Oreochromis niloticus), and Megalobrama amblycephala. Amplification specificity and sensitivity were better than existing methods. The limit of detection for standard PCR was 2.67 theronts/μL, while real-time PCR could be detected at 0.02 theront/μL. Among them, the detection sensitivity of real-time PCR was higher than that of standard PCR. In the detection application of clinical samples and environmental water samples, the two PCR assays showed high consistency, and could effectively detect the latently infected fish and I. multifiliis in the pond water samples. Therefore, the assays developed in this study were suitable tools for early diagnosis and pathogen monitoring of I. multifiliis in freshwater aquaculture.