In situ and directly imaging mitochondria in tissues instead of isolated cells can offer more native and accurate information. Particularly, in the clinical diagnose of mitochondrial diseases such as mitochondrial myopathy, it is a routine examination item to directly observe mitochondrial morphology and number in muscle tissues from patients. However, it is still a challenging task because the selectivity of available probes is inadequate for exclusively tissue imaging. Inspired by the chemical structure of amphiphilic phospholipids in mitochondrial inner membrane, we synthesized a phospholipid-biomimetic amphiphilic fluorescent probe (Mito-MOI) by modifying a C18-alkyl chain to the lipophilic side of carbazole-indolenine cation. Thus, the phospholipid-like Mito-MOI locates at mitochondrial inner membrane through electrostatic interaction between its cation and inner membrane negative charge. Simultaneously, the C18-alkyl chain, as the second targeting group, is deeply embedded into the hydrophobic region of inner membrane through hydrophobic interaction. Therefore, the dual targeting groups (cation and C18-alkyl chain) actually endow Mito-MOI with ultrahigh selectivity. As expected, high-resolution microscopic photos showed that Mito-MOI indeed stained mitochondrial inner membrane. Moreover, in situ and high-fidelity tissue imaging has been achieved, and particularly, four kinds of mitochondria and their crystal-like structure in muscle tissues were visualized clearly. Finally, the dynamic process of mitochondrial fission in living cells has been shown. The strategy employing dual targeting groups should have reference value for designing fluorescent probes with ultrahigh selectivity to various intracellular membranous components.