In 2022, the total freshwater fish production in China reached 2 800.31 million t, accounting for 77.04% of the total domestic fish production. In recent years, against the background of the rapid growth of the ready-to-eat leisure food market in China, a variety of categories of ready-to-eat freshwater fish products have been developed, among which vacuum-packed seasoned fish pieces are one of the mainstream types. However, the processing technology of vacuum-packed seasoned fish pieces still has key technical difficulties, which hinder the development of the industry. The hard fish bones may puncture the packaging bag due to compression during transportation, leading to oil leakage and spoilage, which is the main reason for the high product wastage rate. The presence of large and hard fish bones in the products poses serious food safety hazards, including puncture of the esophagus and damage to organs. The problem of fish bone softening in freshwater fish products needs to be addressed urgently, both from the perspective of production, storage, and transport of the product and from the perspective of food safety. To solve the problem of fish bone softening in ready-to-eat freshwater fish products, the study designed a fish bone softening rate measuring method for ready-to-eat freshwater fish products and then developed the ultrasound-cetic acid (U-CA) fish bone softening technique. The optimal fish bone softening conditions were screened according to the fish bone softening rate and the quality indexes of the fish pieces, and the mechanism of fish bone softening was investigated subsequently. Ctenopharyngodon idella pieces were prepared by ultrasonic combined with acetic acid treatment, marination, deep-frying, vacuum packaging, and autoclaving to obtain soft bone fish pieces, whose quality was evaluated by sensory characteristics, pH value, color value, and muscle structure. The fish bone softening rate measuring method was established according to the sensory evaluation and TPA analysis of the standard fish bone samples. The optimal process conditions of U-CA treatment were obtained by evaluating the bone softening rate. Finally, SEM and FTIR were used to analyze the mechanism of fish bone softening of U-CA treatment. The results showed that the fishbone softening rate measuring method we designed can easily and objectively evaluate the degree of fish bone softening in ready-to-eat fish products. After the evaluation of the bone softening rate, sensory evaluation, and other quality indexes, the optimal U-CA treatment conditions were: acetic acid concentration at 1.2% (V/V), ultrasonic power at 120 W, and processing time of 45 min. The fish pieces obtained under these conditions had better bone softening rate, color, texture, and taste. The results of SEM and FTIR showed that U-CA treatment under the optimal process conditions could weaken the bind between collagen and hydroxyapatite, and contribute to the escape of hydroxyapatite. Heat treatment may also disrupt the triple-helical structure of collagen and reduce the hardness and toughness of fish bones. It was shown that U-CA treatment under appropriate conditions could disrupt the original structure of fish bones and effectively soften the fish bones in C. idella pieces without affecting the quality of the product. This study provides ideas for the development of freshwater fish bone softening technology and theoretical support for the development of ready-to-eat fish.