Detection of bovine serum albumin (BSA) is an important issue in the sense of medical applications and enzymatic reactions; however, the recently developed fluorescent probes require the involvement of dimethyl sulfoxide (DMSO), which may be detrimental to proteins. In this study, we demonstrated a DMSO-free and water-soluble fluorescent probe prepared by ionic co-assembly of amphiphiles. The cationic amphiphile is a newly designed molecule (denoted by DPP-12) bearing a conjugated diketopyrrolopyrrole (DPP) and two tetraphenylethylene groups. It turns out that the fluorescence emission of DPP-12 depends on the amount of anionic amphiphilic sodium dodecyl benzene sulfonate (SDBS). The fluorescence intensity first increases and then decreases with the concentration of SDBS, and each branch presents a linear relationship. BSA consumes SDBS by the formation of complexes, thus leading to an increase of fluorescence intensity of the mixed solution of DPP-12 and SDBS. Therefore, the mixed solution of DPP-12 and SDBS was applied as a fluorescent probe to detect the low concentration of BSA by back-titration. This fluorescent probe does not require DMSO and has good tolerance to metal ions in blood and good photostability. The limit of detection is as low as 940 nM, almost 3 orders of magnitude lower than the content in organisms.